JP2020532969A - Chimeric antigen receptor and CAR-T cells that bind to CXCR5 - Google Patents

Abstract

The present invention relates to an isolated chimeric antigen receptor (CAR) polypeptide, which is an extracellular antigen containing an antibody or antibody fragment that binds to a human CXC chemokine receptor type 5 (CXCR5) protein. Contains a binding domain. The present invention further comprises a nucleic acid molecule encoding the CAR of the invention, an immune cell (preferably a T cell) whose gene has been modified to express the CAR of the invention, and a pathogenic cell expressing CXCR5 (preferably). Medical disorders associated with the presence of pathogenic mature B cells and / or memory B cells, and / or pathogenic T cells and / or T follicular helper cells (particularly mature B cells non-Hodgkin's lymphoma) B-NHL), T-cell non-Hodgkin's lymphoma, autoantibody-dependent autoimmune disease (preferably selected from systemic erythematosus (SLE) and rheumatoid arthritis)) relating to its use in the treatment of cells.

Description

The present invention relates to an isolated chimeric antigen receptor (CAR) polypeptide, which CAR is an extracellular antigen binding comprising an antibody or antibody fragment that binds to the CXC chemokine receptor type 5 (CXCR5) protein. Contains the domain. The present invention further comprises a nucleic acid molecule encoding the CAR of the invention, an immune cell (preferably a T cell) whose gene has been modified to express the CAR of the invention, and a pathogenic cell expressing CXCR5 (preferably). Medical disorders associated with the presence of pathogenic mature B cells and / or memory B cells, and / or pathogenic T cells and / or T follicular helper cells (particularly mature B cells non-Hodgkin's lymphoma) B-NHL), T-cell non-Hodgkin's lymphoma, autoantibody-dependent autoimmune disease (preferably selected from systemic erythematosus (SLE) and rheumatoid arthritis)) relating to its use in the treatment of cells.

B-NHL is diverse and can be distinguished by medium and low grades. Standard therapy is typically an antibody / chemotherapy combination, which is applied alone or in combination with autologous stem cell transplantation, immunomodulators, radiation, proteasome inhibitors, signaling pathway inhibitors. Allogeneic stem cell transplantation is applied to a very small number of patients. Many B-NHL patients are older than the median age of 66-72 at diagnosis, so if they have multiple illnesses, they may not be able to receive intensive chemotherapy, long-term chemotherapy, or allogeneic. Sometimes even bone marrow transplantation is not possible.

Inhibitors of B cell receptor (BCR) signaling in mature B cell lymphomas, especially ibrutinib, have resulted in a significant improvement in remission rates. Despite the initial sensitivity of these classes of kinase inhibitors, it is uncertain whether tumor eradication can be achieved, and in some studies, Breton's evolution of clonal lymphoma and leukemia It has been shown that resistance to tyrosine kinase (BTK) inhibition develops. Therefore, due to the rapid development of secondary resistance to targeted therapy, tolerability applicable to patients who have received several other chemotherapy and therefore have diminished clinical efficacy (IPI score). There is an urgent need to find a solution that can be rescued.

Adopted chimeric antigen receptor (CAR) -T therapy targeting the CD19 antigen, which is widely expressed on the surface of B cells in leukemia and lymphoma, has had great clinical effects, and at present, B-NHL and B- More than 40 CD19 CAR T cell studies aimed at treating ALL have been registered with the FDA (www-clinical trials.gov). However, with anti-CD19 antibody or CAR-T cell therapy against B-NHL, resistance can develop due to antigen loss. A recent study showed that anti-CD19 CAR-T cell therapy selected alternative spliced CD19 isoforms, thus resulting in the appearance of escape variants as a result of the loss of the cognate CD19 CAR epitope.

Therefore, CXCR5 appears as an alternative target for immunotherapy of B-cell lymphoma, in addition to the existing therapeutic 耀 mAb and CAR-T cell therapy.

B-cell-derived lymphoproliferative disorders that are clearly present in the lymph nodes (acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma ( MCL), diffuse large B-cell lymphoma (DLBCL, etc.)) often constitutively express the chemokine receptor CXCR5. CXCR5 is physiologically expressed on the surface of mature recirculating B cells and on the surface of follicular T helper cells (Tfh), a small subset of CD4 ⁺ T cells, which are within secondary lymphoid organs. Adjusts the B cell follicles for constant troughing and homing. Importantly, CXCR5 is not expressed on the surface of B cell precursors in bone marrow (BM), and plasma cells do not express this receptor either.

As far as the inventor knows, no alternative anti-CXCR5 CAR construct has been previously reported, and no studies of anti-CXCR5 antibodies related to the medical approach of the invention are currently known.

Panjihe et al. (International Journal of Cancer, Vol. 135, No. 11, April 29, 2014) describe the treatment of non-Hodgkin's lymphoma using CXCR5 bispecific antibodies. Saderain et al. (Cancer Discovery, Vol. 3, No. 4, April 1, 2013) provide a review of various CAR technologies, but no mention of CXCR5. WO 2016/090034 discloses a number of possible targets for CAR constructs. Although CD185 (CXCR5) is mentioned, there is no detailed description of the components of CAR, medical use, and no mention of the importance of this target. WO 2016/164731 describes the use of CAR-T cells towards a variety of B cell target antigens. No mention is made of CXCR5 as a target for CAR T.

Alternative therapies for the above medical conditions are under development or recently established. For example, anti-CD19 CAR constructs, standard therapies (such as cytotoxic chemotherapy), corticosteroids, immunomodulators (such as IMID), proteasome inhibitors, autologous stem cell transplants, allogeneic stem cell transplants, signaling inhibitors, etc. It is a bispecific antibody (BITE) (blinatumomab) consisting of an antibody against CD20 (rituximab), an anti-CD19 (oletuzumab), a Fab fragment targeting CD19 and an anti-CD3 fragment.

Numerous alternative therapies are under development for pathogenic B-cell and pathogenic T-cell disorders, but it remains to provide effective means of addressing such medical disorders. Greatly needed.

Given the prior art, the technical issues behind the present invention are agents suitable for the treatment of pathogenic B cell and / or T cell related diseases, especially non-Hodgkin's lymphoma or autoantibody-dependent autoimmune diseases. Was to prepare.

This problem is solved by the characteristics of the independent claims. Preferred embodiments of the present invention are provided by the dependent claims.

Therefore, the present invention
i. An extracellular antigen-binding domain containing an antibody or antibody fragment that binds to the CXC chemokine receptor type 5 (CXCR5) protein.
ii. Transmembrane domain and
iii. For chimeric antigen receptor (CAR) polypeptides containing the intracellular domain.

The invention is therefore associated with an anti-CXCR5 CAR construct and the corresponding immune cells expressing this construct, which are normal hematopoietic cells (T cells (Tf lymphomas and other T cells described herein and other T cells). It is preferably a CAR-T cell product that gives human T cells a large cytotoxic activity against a given mature B-NHL without affecting cell lymphoma), trait B cells, their bone marrow precursors, etc.). .. In a preferred embodiment, all bone marrow cells and NK cells are similarly unaffected. This is because the CAR-T cell products of the present invention do not show activity on these cells.

In a preferred embodiment of immunotherapy according to the invention, patient-derived T cells are preferably transduced with a retrovirus to express the artificial immune receptors described herein. This artificial immunoreceptor consists of an extracellular antibody-derived antigen recognition moiety fused to a transmembrane domain, followed by an intracellular signaling domain. Thus, the constructs described herein confer antitumor cell lysing capacity on transduced T cells.

Because of this preferred autotransplantation of T cells, graft-versus-host disease cannot occur when treated with CAR-T of the present invention. It can promote the formation of memory T cells, which are important for the prevention of recurrence.

Non-limiting examples of such mature B-NHL include several stages of follicular lymphoma, diffuse large cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia.

For the first time, anti-CXCR5 CAR cells will be able to target tumor cells in the tumor microenvironment. This is because Tfh cells, which promote the growth of lymphoma, will be accompanied and eradicated. Tumor cells within a given tumor are uniformly positive for the target antigen CXCR5, thus avoiding unwanted positive selection of low / non-expressing tumor cells.

The anti-CXCR5 CAR cells described herein can be applied in a preferred embodiment to the treatment of mature B-NHL patients who are not eligible for other therapies. More specifically, embodiments of the present invention relate to the treatment of the following patient populations:
i) Patients with multidrug resistance and / or ii) Patients who are not eligible for allogeneic stem cell transplantation, and / or iii) Patients with multiple illnesses who cannot receive further chemotherapy, and / or iv) Chemistry Elderly patients who cannot tolerate therapy, and / or v) CAR can be applied for rescue therapy after the disease has progressed or even if some other standard therapy fails, and / or vi) The antibody may not work if the antigen on the target tumor cell is low in density, but CAR is still applicable, and / or not to vi) antibody, but CAR can be applied as a monotherapy.

The anti-CXCR5 CARs described herein provide T cells with the large avidity required for antitumor effects. The anti-CXCR5 CAR of the present invention provides T cell reactivity to physiological trait B cells, T cells (excluding Tfh cells and special pathogenic CXCR5-expressing T cells), NK cells, all bone marrow cell lineages and their precursors. It was proved not to give. Therefore, the present invention has unprecedentedly low off-target reactivity to other hematopoietic tissues.

The anti-CXCR5 CARs of the present invention, unlike anti-CD19 CAR-T cells, do not have the undesired reactivity to immature B-NHL, precursor B cell neoplasms, and physiologically benign B cell precursors.

As demonstrated in the examples below, in in vitro co-culture systems, anti-CXCR5 CAR-T cells are activated upon exposure to CXCR5-expressing human B-NHL tumor cell lines. These T cells then develop an effector phenotype with high levels of IFNγ secretion, a phenotype that predicts cytotoxic activity.

In addition, selective cell injury from cytotoxicity assays ( ⁵¹ Cr release) on B-NHL cells, B cell leukemia cells, T cell leukemia cells, CXCR5-negative cells, and CXCR5 transfectants selected as target cell lines. It can be seen that sex is only obtained in cell lines that are positive for CXCR5.

Additional preclinical studies include i) in vitro cytotoxicity studies of appropriate B-NHL cell lines from patients and ii) in vivo studies of anti-CXCR5 CAR activity on xenograft B-NHL cell lines.

As such, the CARs of the present invention present a phenomenal and beneficial approach to the treatment of the medical conditions described herein. The use of anti-CXCR5 CAR has never been attempted or reported as a promising approach to the treatment of NHL. Minimal, if not nonexistent, unwanted side effects due to marker selectivity are also beneficial and surprising aspects of the invention. The present invention provides a very promising approach for the eradication of malignant tumors, especially in patients who have developed resistance to anti-CD19 therapy.

In one embodiment, the invention relates to the chimeric antigen receptor (CAR) polypeptide described herein, wherein the CAR is present in a genetically modified immune cell (preferably T lymphocyte). When expressed in, the immune cell induces cytotoxic activity against the cell expressing CXCR5 by binding to and activating CXCR5 on the surface of the cell expressing CXCR5.

Examples of cells expressing CXCR5 are known to those of skill in the art and can be identified by further screening of cancer and other pathogenic cells. The cell lines expressing CXCR5 are the DOHH-2 cell line and / or the OCI-Ly7 cell line and / or the SU-DHL4 cell line and / or the JeKo-1 cell line and / or the JVM-3 cell line. And / or MEC-1 cell lines and / or SC-1 cell lines are preferred.

In one embodiment, the invention relates to a chimeric antigen receptor (CAR) polypeptide, which CAR includes.
-An extracellular antigen-binding domain containing an antibody or antibody fragment that binds to the CXC chemokine receptor type 5 (CXCR5) protein (although the antibody or antibody fragment contains the VH and VL domains of a single chain antibody fragment and is a linker polypeptide. Is preferably located between the VH domain and the VL domain, and the linker is preferably located so as not to interfere with the interaction between the antibody fragment and the CXCR5 antigen).
-A spacer polypeptide (also called a hinge) located between the extracellular antigen-binding domain and the transmembrane domain (although this spacer polypeptide expresses a configuration that does not interfere with the interaction of the antibody fragment with the CXCR5 antigen and / or CAR. It is preferable that the arrangement does not interfere with the activation of the T cell when the CAR is expressed in the T cell.
-Transmembrane domain (although this transmembrane domain is located in a configuration that does not interfere with the interaction of the antibody fragment with the CXCR5 antigen and / or activates the T cell when the CAR is expressed in the T cell expressing the CAR. It is preferable that the arrangement does not interfere) and;
-The intracellular domain (this intracellular domain contains a co-stimulation domain and a signal transduction domain, and activates T cells when bound to a CXCR5 target, for example by increasing cytokine production and / or promoting T cell replication. It is preferably an arrangement that provides a cytotoxic effect by providing a stimulating signal).

Accordingly, the CARs of the present invention can take various forms, including protein sequences that may differ from each other in their functional domains as described herein. One of ordinary skill in the art can select and test the desired function of CAR based on, for example, the experimental approach shown in the examples below. As such, for any of the functional domains discussed herein, any specific protein sequence selected for use in the CAR of the present invention will be functionally functional by one of ordinary skill in the art using routine methods. The effect of can be evaluated. For example, various linker polypeptide sequences located between the VH and VL domains, various spacer polypeptide sequences (also called hinges) located between the extracellular antigen-binding domain and the transmembrane domain, and various transmembrane domains. A variety of intracellular domains (preferably containing a co-stimulation domain and a signaling domain) can be used.

In embodiments of the invention, the CAR and each of the elements or domains referred to herein are arranged in a T cell expressing CAR in a non-harmful arrangement for the interaction of the antibody fragment with the CXCR5 antigen. Arranged so that it does not interfere harmfully with T cell activation when the CAR is expressed, and does not interfere harmfully with CAR that provides a signal that stimulates T cell activation when bound to a CXCR5 target. There is.

Experimental methods for assessing these properties of CXCR5 CAR are described herein, and the present invention combines various functional sequence variants with the types of domains described herein. Although considered to be included, inclusion is not limited to the particular sequences disclosed below as examples. For example, specific activation of CAR-T cells of the invention by tumor cells expressing CXCR5 can be demonstrated by the release of IFNγ, IL-2, TNF-α, as shown below. ..

As far as the inventor knows, the present invention relates to the first described CXCR5 CAR and evidence that the desired therapeutic effect of the CXCR5 CAR worked for the first time in the medical setting. Independent of the individual sequences used in the various functional domains described herein, the CXCR5 CAR simply provides pathogenic mature B cells and / or memory B cells, and / or pathogenic T cells. It provides significant and beneficial breakthroughs in the treatment of many diseases associated with cells and / or T follicular helper cells.

Drawing

The present invention will be clarified by the following drawings as an example. The drawings should be considered to provide a more detailed description of potentially preferred embodiments that support one or more non-limiting embodiments of the present invention.

The schematic diagram of the structure of a preferable CAR.

Schematic of the preferred CAR constructs H28, R28, HBB1, HBB2, H28BB.

A list of possible combinations of the preferred CAR constructs described herein and the various structural elements of CAR.

Sequence comparison between the mAb binding region and the preferred humanized sequence used in the CAR of the present invention.

A sequence diagram of a DNA sequence encoding the mAb binding region hHC.

A sequence diagram of a DNA sequence encoding the mAb binding region hLC.

A GeneArt ™ plasmid with a humanized CXCR5-CAR sequence and a GeneArt ™ plasmid with a rat CXCR5-CAR sequence. A GeneArt ™ plasmid with a humanized CXCR5-CAR sequence and a GeneArt ™ plasmid with a rat CXCR5-CAR sequence.

Gel electrophoresis of constructs and vectors after restriction. Gel electrophoresis of constructs and vectors after restriction.

Confirmation by flow cytometry that CXCR5-CAR is expressed on the surface of human T cells after transduction using a retrovirus.

Expression of CXCR5 on the surface of various types of cells evaluated in the functional assay. Expression of CXCR5 on the surface of various types of cells evaluated in the functional assay.

Co-culture of CAR transduced human T cells with various target cell lines reveals specific T cell activation by distinguishable CXCR5 ⁺ B-NHL and control cell lines.

Cytotoxicity assays reveal selective killing of CXCR5-positive cell lines. The cytotoxic assay reveals selective killing of CXCR5-positive cell lines.

CXCR5-redirected CAR-T cells are effective against B-cell non-Hodgkin's lymphoma (B-NHL) in xenografted NSG mouse models. CXCR5-redirected CAR-T cells are effective against B-cell non-Hodgkin's lymphoma (B-NHL) in xenografted NSG mouse models.

Co-culture of CAR transduced human T cells with various target cell lines reveals specific T cell activation by distinguishable CXCR5 ⁺ B-NHL and control cell lines. Co-culture of CAR transduced human T cells with various target cell lines reveals specific T cell activation by distinguishable CXCR5 ⁺ B-NHL and control cell lines.

Co-culture of CAR-transduced human T cells with CXCR5-negative primary cells from various target human tissues reveals no off-target T cell activation, whereas B- expressing CXCR5. The NHL cell line JeKo-1 mediates specific T cell activation and functions as a positive control.

Embodiments relating to the antigen binding domain of CAR:

In one embodiment, the invention relates to the chimeric antigen receptor (CAR) polypeptide described herein, wherein the antigen binding domain thereof.
-Heavy chain complementarity determining regions 1 (H-CDR1), whose sequence matches at least 80% with SEQ ID NO: 1 (GFTFSTSG),
-Heavy strand complementarity determining regions 2 (H-CDR2), whose sequence matches at least 80% with SEQ ID NO: 2 (ISSSSGFV),
-A variable heavy chain (VH) containing heavy chain complementarity determining regions 3 (H-CDR3) whose sequence is at least 80% identical to SEQ ID NO: 3 (ARSEAAF).
-Light chain complementarity determining regions 1 (L-CDR1), whose sequence is at least 80% identical to SEQ ID NO: 4 (KSRLSRMGITP),
-Light chain complementarity determining regions 2 (L-CDR2), whose sequence is at least 80% identical to SEQ ID NO: 5 (RMS),
-Contains a variable light chain (VL) containing light chain complementarity determining regions 3 (L-CDR3) whose sequence is at least 80% identical to SEQ ID NO: 6 (AQFLEYPPT).

In one embodiment, the invention relates to the chimeric antigen receptor (CAR) polypeptide described herein, which CAR comprises the CDR sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. It contains a VH domain and a VL domain containing the CDR sequences of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

In a preferred embodiment, sequence variants whose sequences match 80% or more of the specific CDR sequences of SEQ ID NOs: 1-6 are substantially the VH and VL domains having the specific CDR sequences of SEQ ID NOs: 1-6. It maintains binding to CXCR5 with the same or similar functional properties. That is, binding to CXCR5 is substantially the same or similar in terms of affinity, specificity, and epitope binding mode.

The amino acid sequence of the scFV fragment was originally obtained from a rat anti-human CXCR5 antibody, for example, by making many improvements by humanizing the VL and VH chains, the context of the transmembrane receptor structure. Folding and manifestation in.

In addition, the order of the light chain fragment and the heavy chain fragment can be reversed taking into account the desired arrangement of the antigen binding fragment.

In addition, in some embodiments, the CAR function is enhanced by modifying the linker sequence between the heavy and light chains, for example by shortening.

In addition, the expression of CAR is improved by optimizing the codons of the nucleic acid sequence encoding CAR.

These modifications allow for full expression on the surface of T cells and maintain proper antigen binding as ever. Due to its large affinity and large avidity, CAR-T cells recognize i) tumor target cells with high, medium and low expression of CXCR5 on the surface, and ii) are activated against the tumor target cells. , Iii) The tumor target cells can be killed.

As far as the inventor knows, neither anti-CXCR5 CAR nor humanized anti-CXCR5 antibody has been reported in the art.

Due to the high affinity and avidity of the antigen-binding domains of the anti-CXCR5 CAR cells described herein, even mature B-NHL with low expression of CXCR5 can be recognized, T cell activation and tumor cell activation. Killing is possible.

The anti-CXCR5 CAR cell products described herein are characterized by unique properties.

The anti-CXCR5 CARs described herein have a great affinity and provide great specificity and avidity for T cells. Due to these properties, CAR-T cells recognize i) tumor target cells with high CXCR5 and low tumor target cells expressed on the surface, ii) activated against the tumor target cells, and iii) its. It becomes possible to kill tumor target cells.

The number of CXCR5 antigens expressed on the surface of tumor cells can be quantified by an anti-CXCR5 antibody coupled to a fluorescent dye in combination with Quantibrite beads (from Becton Dicksinson). A preferred method applied to the quantification of CXCR5 antigen expressed on the surface of tumor cells is "fluorescence activated cell sorting / cell analysis" (FACS). This is one indicator of the number of CXCR5 molecules on the surface of the cell, as the fluorescence intensity of the beads correlates exactly with the number of fluorescent antibodies bound to the cell.

Although the VH and VL fragments described herein can be located in multiple arrangements within the CAR, they still maintain great specificity and high affinity for the target epitope. In some embodiments, the CAR can be in a VH-VL or VL-VH configuration, a linker and / or a hinge, and / or a transmembrane domain, and / or a costimulatory domain, and / or. There are variations in the activation domain, but the effect is still maintained. If there is a need or desire for further development, this amazing feature of the invention allows for greater flexibility in the design of the CAR towards the CXCR5, thereby increasing the structure of the CAR. Can be further modified and / or optimized based on the VH and VL domains described herein.

The sequence alignment of the rat and humanized sequences is shown in FIG. Thus, the sequences below also include generalized sequences representing antigen-binding domains in both rat and humanized forms. Each X in the sequence below indicates that the amino acid may change. Preferred amino acid substitutions are the amino acid substitutions described for each position that may change.

For any given variant residue (specified by X in the "generalized sequence") that may change as proposed herein, any possible modificationable combination is available. Included in the present invention. Combining one or more of these various substitutions can produce a humanized variant that exhibits the desired binding injury of the rat antigen binding domain demonstrated herein. CAR or a portion thereof described herein also includes sequences that are at least 70%, 80%, preferably 90% consistent with the humanized sequence disclosed explicitly, i.e., in the formula for one sequence. included.

In one embodiment, the invention relates to the chimeric antigen receptor (CAR) polypeptide described herein, wherein the CAR is SEQ ID NO: 7:
(However, X1 is G or K; X2 is R or K; X3 is A or S; X4 is N or H; X5 is E or D; X6 is S or A; X7 is S or A; X8 is S or T; X9 is M or L; X10 is R or K; X11 is A or S; X12 is V or I)
And a VH domain whose sequence matches at least 70% or 80%, preferably at least 85%, or 90%, or 95%, or 100%.
SEQ ID NO: 8:
(However, X1 is S or A; X2 is L or V; X3 is P or S; X4 is S or N; X5 is Q or K; X6 is R or L; X7 is G or E)
Includes a VL domain whose sequence is at least 80% matched, preferably at least 85%, or 90%, or 95%, or 100% matched.

In a preferred embodiment, the specific VH sequences and VL sequences described herein and sequence variants that are 80% or more identical in sequence are substantially the same as the VH and VL domains having the specific sequences described herein. Maintains binding to CXCR5 with the same or similar functional properties. That is, binding to CXCR5 is substantially the same or similar in terms of affinity, specificity, and epitope binding mode.

In one embodiment, the invention relates to the chimeric antigen receptor (CAR) polypeptide described herein, which CAR is:
A VH domain according to SEQ ID NO: 9 (EVQLVESGGGLVQPGGSLRLSCAASGFTFSTSGMNWFRQAPGKGLEWVSYISSSSGFVYADSVKGRFTISRDNAQNSLYLQMNSLRAEDTAVYYCARSEAAFWGQGTLVTVSS), or SEQ ID NO: 10 (EVQLVESGGGLVQPGKSLKLSCSASGFTFSTSGMHWFRQAPGKGLDWVAYISSSSGFVYADAVKGRFTISRDNAQNTLYLQLNSLKSEDTAIYYCARSEAAFWGQGTLVTVSS),
Containing a VL domain according to SEQ ID NO: 11 (DIVLTQSPRSLPVTPGEPASISCRSSKSRLSRMGITPLNWYLQKPGQSPQLLIYRMSNRASGVPDRFSGSGSGTDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK), or SEQ ID NO: 12 (DIVLTQAPRSVSVTPGESASISCRSNKSRLSRMGITPLNWYLQKPGKSPQLLIYRMSNLASGVPDRFSGSGSETDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK).

In yet another embodiment, the invention relates to a chimeric antigen receptor (CAR) polypeptide comprising one or more linkers, spacers, transmembrane domains, signaling domains. In one embodiment, the CAR comprises an intracellular domain that includes a co-stimulating domain and a signaling (activating) domain.

In one embodiment, the invention relates to the chimeric antigen receptor (CAR) polypeptide described herein, wherein the extracellular antigen binding domain is located between the VH domain and the VL domain. The choice of this linker contains a linker polypeptide,
-Whitelinker (SEQ ID NO: 13: GSTSGSGGKPGSGEGSTKG),
-Gly-Ser linker (SEQ ID NO: 14: SSGGGGGSGGGGSGGGGS),
-Preferably made from a linker whose sequence matches at least 80% with SEQ ID NO: 13 or 14.

In one embodiment, the invention relates to the chimeric antigen receptor (CAR) polypeptide described herein, which CAR adds a spacer polypeptide between the extracellular antigen binding domain and the transmembrane domain. The selection of this spacer includes
- IgG1 spacer (SEQ ID NO: 15; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK)
- IgG1deruta spacer (SEQ ID NO: 16; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSSLSPGKK)
- IgG4 (Hi-CH2-CH3) spacer (SEQ ID NO: 17; ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK);
-IgG4 (Hi-CH3) spacer (SEQ ID NO: 18; ESKYGPPPCPPCPGQPREPQVYTLPPPSQEEMTKKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCVSVMSHESLVSHLSLHKS
-IgG4 (Hi) spacer (SEQ ID NO: 19; ESKYGPPCPPCP);
-Made from spacers whose sequence matches at least 80% with any one of SEQ ID NOs: 15-19.

In one embodiment, the invention relates to the chimeric antigen receptor (CAR) polypeptide described herein, the selection of a transmembrane domain therein.
-CD8α domain (SEQ ID NO: 20; IYIWAPLAGTCGVLLLSLVITLYC),
-CD28 domain (SEQ ID NO: 21; FWVLVVVGVGVLACYSLLVTVAFIIFWV),
-Made from a transmembrane domain whose sequence matches at least 80% with SEQ ID NO: 20 or 21.

In one embodiment, the invention relates to the chimeric antigen receptor (CAR) polypeptide described herein, in which the intracellular domain comprises.
− 4-1BB co-stimulation domain (SEQ ID NO: 22; KRGRKKKLLYIFKQPMFMRPVQTTQEEDGCSCRFPEEEEGGCEL) and / or − CD28 co-stimulation domain (SEQ ID NO: 23; RSKRSRLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAYRSL) (SEQ ID NO: 23; ) In an adjacent arrangement, or a co-stimulation domain selected from a co-stimulation domain whose sequence matches at least 80% with SEQ ID NO: 22 or 23.

In one embodiment, the invention relates to the chimeric antigen receptor (CAR) polypeptide described herein, which CAR adds a signaling domain (also known as an activation domain). Contains and this signaling domain
− CD3ζ (4-1BB or CD28) signaling domain (SEQ ID NO: 24; LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRGRDPEMGGRGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRGKGHDGLYNELQKDKMAEAYSEIGMKGERRGKGHD

In one embodiment, the invention relates to the chimeric antigen receptor (CAR) polypeptide described herein, wherein the CAR is any one of SEQ ID NOs: 25, 26, 27, 28, 29. Contains sequences that follow.

The exchange of signaling domains meets the requirement for long-lasting recurrence control guaranteed by a strong and rapid effector phase (CD28 co-stimulating domain), or T cell memory population (4-1BB signaling domain). As demonstrated herein, the various signaling domains can be exchanged for a large number of configurations to provide a design-flexible CAR without losing favorable binding properties. ..

In yet another embodiment of the invention, the CAR has the following arrangement:
-H28: MP71-hCXCR5-VH-White-VL-IgG1-CD28-CD28-CD3z
-R28: MP71-Rat CXCR5-VH-White-VL-IgG1-CD28-CD28-CD3z
− HBB1: MP71-hCXCR5-VH-White-VL-IgG1Δ-CD8α-4-1BB-CD3z
− HBB2: MP71-hCXCR5-VH-White-VL-IgG1-CD28-4-1BB-CD3z
− H28BB: MP71-hCXCR5-VH-White-VL-IgG1-CD28-CD28-4-1BB-CD3z
Can be included.

These special arrangements are preferred, but not limited to. The above arrangement is not limited by the specific sequences of the embodiments described herein. Variations in the arrangement are possible within the context of these arrangements, and the variations are within the scope of the present invention.

Variants used as linkers, spacers, transmembrane domains, and intracellular domains (by adding alternative components) allow those skilled in the art to replace various components as needed to bind properties to CXCR5. It is clear that the desired biological effect can be maintained by maintaining.

In a preferred embodiment, an abnormally large transduction rate can be achieved in human T cells when combined with an MP71-vector and a γ-retrovirus expression system. Transduction systems vary because the CAR construct has a modular design. This means that lentiviruses and transposons can be used according to the needs and preferences of those skilled in the art when carrying out the present invention. For the introduction of genetic information / nucleic acid molecules for CXCR5 CAR, insert into target cell lines (preferably T lymphocytes, natural killer cells, induced pluripotent stem cells (iPS)) via Crisp / Cas and TALEN. Is also included. Any method suitable for introducing the genetic information / nucleic acid molecule for CXCR5 CAR into cells expressing that CAR is included in the present invention, and suitable methods will be practiced by those skilled in the art. Can be selected when For example, a number of methods for transforming T cells are known in the art, including virus-based gene transfer methods (such as modified retrovirus-based methods) and non-viral methods (in DNA). Includes transposons based on, direct introduction of mRNA by electroporation).

In addition, the signaling components of the CAR construct are exchanged in a modular composition and a three-step cloning procedure that allows expert clinically applicable configurations of anti-CXCR5 CAR.

In yet another aspect of the invention, the invention relates to an isolated nucleic acid molecule, which isolated nucleic acid molecule in the form of a vector (eg, in the form of a viral vector or a transposon vector). It is preferably a sleeping beauty vector, and the selection thereof is
a) -A nucleotide sequence encoding the chimeric antigen receptor (CAR) polypeptide described herein, or-an extracellular antigen that encodes an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain. A nucleotide sequence in which the binding domain is encoded by at least one of SEQ ID NOs: 37, 38 and at least one of SEQ ID NOs: 39, 40, or-SEQ ID NOs: 31, 32, 33, 34, Nucleic acid molecule containing a nucleotide sequence according to any of 35;
b) Nucleic acid molecule complementary to the nucleotide sequence according to a);
c) The sequences are matched to such an extent that they have similar / equivalent functions to the nucleotide sequences described in a) or b), preferably at least the nucleotide sequences and sequences described in a) or b). Nucleic acid molecule containing a nucleotide sequence that matches 80%;
d) Nucleic acid molecules that, as a result of the genetic code, become the nucleotide sequences described in a) to c) due to decompression;
e) Modified by deletions and / or additions and / or substitutions, and / or rearrangements, and / or inversions, and / or insertions, but similar in function to the nucleotide sequences described in a) to d). / Equivalent, consisting of a group of nucleic acid molecules according to the nucleotide sequences according to a) to d).

In a preferred embodiment of the invention, the isolated nucleic acid molecule is preferably in the form of a vector (eg, in the form of a viral vector or a transposon vector), preferably a sleeping beauty vector, the choice of which is
a) -A nucleotide sequence encoding the chimeric antigen receptor (CAR) polypeptide described herein, or-an extracellular antigen that encodes an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain. A nucleotide sequence in which the binding domain is encoded by at least one of SEQ ID NOs: 37, 53, 38 and at least one of SEQ ID NOs: 39, 54, 40, or-SEQ ID NOs: 31, 32, Nucleic acid molecule containing a nucleotide sequence according to any of 33, 34, 35;
b) Nucleic acid molecule complementary to the nucleotide sequence according to a);
c) The sequence is matched to such an extent that it has similar / equivalent function to the nucleotide sequence described in a) or b) with respect to binding to the CXCR5 target antigen, preferably described in a) or b). CAR-T cell products are relative to mature B-NHL when they contain a nucleotide sequence that is at least 80% (preferably 90% or 95%) identical in sequence to the nucleotide sequence of the corresponding T cell and expresses the construct. While imparting cytotoxic activity, it does not affect normal hematopoietic cells (excluding T cells and other T cell lymphomas described herein), trait B cells, their bone marrow precursors, etc. Nucleic acid molecule;
d) As a consequence of the genetic code, it consists of a group of nucleic acid molecules that, by reduction, become the nucleotide sequences described in a) to c).

The term degenerate (or degenerate) means that differences in the nucleotide sequence of a nucleic acid molecule do not lead to differences in the amino acids of the protein product of that nucleotide sequence after translation because of the genetic code. In one embodiment, the nucleic acid molecule relates to the molecules of a) and b), or the molecules of a), b) and c), or the molecules of a), b) and d).

Preferred amino acid and nucleotide sequences of the present invention:

Yet another aspect of the invention relates to vectors containing the nucleic acid molecules described herein, which are preferably viral vectors, even more preferably gamma retroviral vectors. In another aspect of the invention, the invention relates to a transposon vector (preferably a sleeping beauty vector) that encodes the CAR of the invention and is preferably capable of expressing that CAR.

Yet another aspect of the invention relates to a genetically modified immune cell comprising a nucleic acid molecule or vector described herein and / or a genetically modified immune cell expressing the CAR described herein.

In a preferred embodiment, the immune cells administered in the treatment of the diseases referred to herein use a "sleeping beauty" transposon system (particularly a sleeping beauty transposase) to provide the anti-CXCR5 CAR described herein. Genes are modified with the nucleic acids described herein that are encoded and expressed. The sleeping beauty transposon system is designed to introduce precisely defined DNA sequences into vertebrate chromosomes for the purpose of modifying immune cells to express the CARs described herein in the context of the present invention. Synthetic DNA transposon. Sleeping beauty transposons combine the benefits of viruses with the benefits of naked DNA. Viruses have been selected and evolved based on their ability to infect new host cells and replicate in them. At the same time, cells have evolved key molecular defense mechanisms to protect themselves from viral infections. It is also important to avoid the use of viruses for social and regulatory reasons. Therefore, the use of non-viral vectors, such as the sleeping beauty system, avoids many, if not all, of the defenses that cells use against the vector. For that reason, the sleeping beauty system allows immune cells to be administered to patients to be genetically modified particularly effectively and safely.

In yet another embodiment of the invention, a nucleic acid encoding CXCR5 CAR can be inserted via CRISPR / Cas and TALEN. Known to those of skill in the art, CrispR / Cas is a modification of a natural process in bacteria that can be used to accurately and effectively edit DNA and obtain appropriate coding sequences for immune cells of interest (preferably). Can be inserted into T cells). Cas9 is a protein that functions as molecular-level scissors and is guided to a specific DNA sequence by the associated RNA molecule (guide RNA). When Cas9 arrives at a target location on DNA, it makes the local genetic code volatile and affects the function of that gene. CRISPR / CAS9 can deliver the CAR gene to a very specific site within the T cell genome. Therefore, the risk of gene insertion at inaccurate or undesired positions can be reduced.

In one embodiment, the immune cells are preferably selected from the group consisting of T lymphocytes and NK cells, more preferably from cytotoxic T lymphocytes.

In a preferred embodiment, immune cells comprising the nucleic acid molecules or vectors described herein, and / or immune cells expressing CAR described herein, are CD4 ⁺ T cells and / or CD8 ⁺ T cells. It is characterized by being, preferably a mixture of CD4 ⁺ T cells and CD8 ⁺ T cells. These T cell populations, preferably compositions containing both CD4 ⁺ transformed cells and CD8 ⁺ transformed cells, are preferably relative to various malignant B cells (such as B-NHL), these cells and / or It exhibits cell lytic activity that is particularly effective against the related medical conditions described herein.

In a preferred embodiment, the genetically modified immune cells comprising the nucleic acid molecules or vectors described herein and / or the genetically modified immune cells expressing the CAR described herein are CD4 ⁺ T cells and CD8 ⁺ T cells. It is a cell, and its ratio is preferably 1:10 to 10: 1, more preferably 5: 1 to 1: 5, or 2: 1 to 1: 2, or 1: 1. The modified CAR-T cells expressing the CARs described herein and heading for CXCR5 are administered in the above ratios (preferably a 1: 1 ratio of CD4 ⁺ : CD8 ⁺ ) as referred to herein. It leads to beneficial features while treating the disease. For example, these ratios improve the response to treatment and reduce toxicity.

One additional surprising aspect of the invention is the improved stability of CAR disclosed herein. Under appropriate conditions, CAR polypeptides can be easily stored for extended periods of time without any loss of binding affinity.

Yet another aspect of the invention relates to the genetically modified immune cells described herein for use in the treatment of medical disorders associated with the presence of pathogenic cells expressing CXCR5.

In one embodiment, the medical disorder to be treated is related to the presence of pathogenic mature B cells and / or memory B cells.

In one embodiment, the medical disorder to be treated is mature B-cell non-Hodgkin's lymphoma (B-NHL).

In one embodiment, the medical disorder to be treated is a B cell-derived lymphoproliferative disorder, acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle. It is preferably selected from the group consisting of cell lymphoma (MCL) and diffuse large B-cell lymphoma (DLBCL).

In one embodiment, the medical disorder to be treated involves pathogenic T cells and / or T follicular helper cells.

In one embodiment, the medical disorder to be treated is T-cell non-Hodgkin's lymphoma with or without leukemic tumor cell dissemination.

In one embodiment, the medical disorder to be treated is T cell-derived lymphoproliferative injury, selected from the group consisting of angioimmunoblastic T-cell lymphoma, cutaneous T-cell lymphoma, and T-cell lymphoma with leukemic dissemination. Is preferable.

Yet another aspect of the invention relates to the genetically modified immune cells described herein expressing the CAR of the invention for use as a drug in the treatment of autoantibody-dependent autoimmune diseases.

In a preferred embodiment, the autoimmune disease is selected from systemic lupus erythematosus (SLE) and rheumatoid arthritis.

Most recently, CAR-T cells have also been discussed as a targeted approach for the treatment of autoantibody-mediated diseases (Ellebrecht et al. (2016) Science Vol. 353: 179- Page 184). The ability to target CXCR5 is thought to be of great benefit for the treatment of autoimmune diseases as it is thought to suppress coexisting autoreactive B and Tfh cells.

Mild forms of autoimmune disease are usually first treated with non-steroidal anti-inflammatory drugs (NSAIDs) or disease-modifying antirheumatic drugs (DMARDs). More severe rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) are usually steroid-resistant immunosuppressants (targeting circulating cells) because organ failure is involved due to the high momentum of the disease. It is treated in combination with cyclophosphamide, which is a cytotoxic agent. Most recently, belimumab, an antibody that targets the cytokine BAFF, which has been found to have elevated levels in the sera of patients with autoimmune diseases, has been approved by the Food and Drug Administration (FDA) for use in SLE.

However, for survival in humans, only newly formed B cells depend on BAFF, while memory B cells and plasma cells are less sensitive to selective BAFF suppression (Jacobi et al. (2010)). Arthritis Rheum Vol. 62: pp. 201-210). For rheumatoid arthritis (RA), TNF inhibitors were the first approved biological agents, followed by abatacept, rituximab, tocilizumab and the like. They suppress important inflammatory pathways involved in joint inflammation and destruction, but at the cost of increased risk of infection due to their relatively low immunity (Chan et al. (2010) Nat Rev Immunol. Volume 10: pp. 301-316, Keyser (2011) Curr Rheumator Rev Volume 7: pp. 77-87).

In particular, rituximab (a monoclonal antibody that eliminates B cells from the circulation) is increasingly prescribed not only for the treatment of RA, but also for the treatment of granulomatosis with polyangiitis and other anti-neutrophil cytoplasmic antibody-related vasculitis. It has become so. However, rituximab is not risk-free and shows similar adverse event rates to cyclophosphamide (Shah et al. (2015) ImmunoTargets and Therapy, Vol. 4, pp. 173-183). Therefore, there is a need for a more finely regulated and longer lasting approach that targets autoreactive B cells and autoantibody reactions.

The present invention further relates to a method of treating a medical condition described herein, which typically comprises a CAR, or an amount effective in treating immune cells expressing the CAR. Includes administration to patients in need of treatment.

Detailed description of the invention
Chimeric antigen receptor:

According to the present invention, the chimeric antigen receptor (CAR) polypeptide comprises an extracellular antigen binding domain containing an antibody or antibody fragment that binds to a CXC chemokine receptor type 5 (CXCR5) protein, a transmembrane domain, and intracellular. Contains the domain. CAR is typically described as including an extracellular external domain (antigen binding domain) derived from an antibody and an internal domain containing a signaling module derived from a T cell signaling protein.

In a preferred embodiment, the external domain preferably comprises variable regions from the heavy and light chains of immunoglobulins configured as single chain variable fragments (scFv). The scFv is preferably bound to the hinge region. This hinge region provides flexibility and transmits signals through transmembrane portions that are anchored to the intracellular signaling domain. The transmembrane domain is preferably derived from CD8α or CD28. In the first generation CAR, the signaling domain consists of the ζ chain of the TCR complex. The term "generation" refers to the structure of the intracellular signaling domain. Second generation CARs have a single co-stimulation domain derived from CD28 or 4-1BB. Third generation CAR already contains two costimulatory domains (eg CD28, 4-1BB, ICOS or OX40, CD3ζ). The present invention preferably relates to 2nd or 3rd generation CARs.

In various embodiments, genetically engineered receptors that redirect the cytotoxicity of immune effector cells to B cells are provided. These genetically engineered receptors are referred to herein as chimeric antigen receptors (CARs). CAR combines antibody-based specificity for the desired antigen (eg, CXCR5) with the intracellular domain that activates the T cell receptor to generate chimeric proteins that exhibit specific anti-CXCR5 cell-mediated immune activity. It is a molecule. As used herein, the term "chimera" describes that it is composed of parts of various proteins or DNAs of different origins.

The CARs considered herein include an extracellular domain (also referred to as a binding domain or antigen binding domain) that binds to CXCR5, a transmembrane domain, and an intracellular or intracellular signaling domain. When the anti-CXCR5 antigen-binding domain of CAR binds to CXCR5 on the surface of target cells, CAR clusters and activation stimuli are transmitted to CAR-containing cells. A key feature of CAR is to redirect the specificity of immune effector cells, which can mediate proliferation, or cytokine production, or fagocytosis, or cell death of target antigen-expressing cells. The production of molecules is initiated in a manner independent of the major histocompatibility complex (MHC) and utilizes the cell-specific targeting ability of monoclonal antibodies, or soluble ligands, or cell-specific co-receptors.

In various embodiments, the CAR comprises a humanized CXCR5-specific binding domain, a transmembrane domain, and one or more intracellular signaling domains. In a particular embodiment, the CAR comprises an extracellular binding domain comprising a humanized anti-CXCR5 antigen binding fragment, one or more spacer domains, a transmembrane domain, and one or more intracellular signaling domains. ..

The "extracellular antigen binding domain" or "extracellular binding domain" is used interchangeably to provide CAR capable of specifically binding to the target antigen of interest, CXCR5. The binding domain can be a natural source, a synthetic source, a semi-synthetic source, or a recombinant source. The scFv domain is preferred.

"Specific binding" is understood in the same sense that one of ordinary skill in the art understands, and one of ordinary skill in the art is clearly aware of the various experimental procedures available for examining binding and binding specificity. .. Methods for determining the equilibrium association constant or the equilibrium dissociation constant are known in the art. For many protein-protein interactions, some cross-reactivity or background binding may be unavoidable, but this does not compromise the "specificity" of binding between CAR and epitope. "Specific binding" describes that an anti-CXCR5 antibody or antigen-binding fragment thereof (or CAR containing it) binds to CXCR5 with greater binding affinity than background binding. The expression "towards" can also be applied when considering the term "specificity" in understanding the interaction between an antibody and an epitope.

"Antigen (Ag)" means a compound, composition or substance capable of stimulating antibody production or T cell response in an animal. In a particular embodiment, the target antigen is an epitope of the CXCR5 polypeptide. "Epitope" means a region of an antigen to which a binder binds. Epitopes can be formed from both continuous and discontinuous amino acids that are adjacently arranged by protein-folded tertiary structure.

A "single chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, which domains are in either orientation (eg, VL-VH) within a single polypeptide chain. Or it exists in VH-VL). In general, scFv polypeptides can further include a polypeptide linker between the VH and VL domains to form the desired structure for antigen binding. In a preferred embodiment, the CAR considered herein comprises an antigen-specific binding domain. It is a scFv and can be either a mouse scFv, a human scFv, or a humanized scFv. Single chain antibodies can be cloned from hybridoma V region genes specific for the desired target. In a particular embodiment, the antigen-specific binding domain is a humanized scFv that binds to a human CXCR5 polypeptide. One example of a variable heavy chain suitable for constructing the anti-CXCR5 CAR considered herein is, but is not limited to, the amino acid sequence set forth in SEQ ID NO: 9. One example of a variable light chain suitable for constructing the anti-CXCR5 CAR considered herein is, but is not limited to, the amino acid sequence set forth in SEQ ID NO: 11.

Antibodies and antibody fragments:

The CAR contains an extracellular antigen binding domain containing an antibody or antibody fragment that binds to the CXCR5 polypeptide. Thus, non-limiting examples of the antibodies or antibody fragments of the invention include polyclonal antibodies, monoclonal antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-stranded fragments (scFv), Single-stranded variable fragment (ssFv), single domain antibody (such as VHH fragment from nanobody), Fab fragment, F (ab') ₂ fragment, fragment prepared by Fab expression library, anti-idiotype antibody, epitope binding fragment , Any combination of these, but retaining the same binding properties as the CAR described herein, preferably containing the corresponding CDR, or VH and VL regions described herein. Is a condition. Mini-antibodies and multi-valent antibodies (divalent antibody, trivalent antibody, 4-valent antibody, 5-valent antibody) can also be used in the method of the present invention. The immunoglobulin molecule of the present invention can be any class (ie, IgG, IgE, IgM, IgD, IgA) or subclass of immunoglobulin molecule. Therefore, the term antibody herein, whether produced by modification of the entire antibody or newly synthesized using recombinant DNA methods, is the antibody and antibody fragment contained in the CAR of the present invention. Also includes.

As used herein, "antibody" generally means a protein consisting of an immunoglobulin gene or one or more polypeptides that is substantially encoded by an immunoglobulin gene or fragment of an immunoglobulin gene. When the term "antibody" is used, it can be considered that the term "antibody fragment" is also referred to. It is recognized that the immunoglobulin genes include constant region genes such as κ, λ, γ, δ, ε, and μ, as well as a large number of immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, ε, which in turn define immunoglobulin classes IgG, IgM, IgA, IgD, IgE, respectively. The basic structural units of immunoglobulins (antibodies) are known to include tetramers or dimers. Each tetramer is composed of two pairs of the same polypeptide chain, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70). It has kD). The N-terminus of each chain defines a variable region consisting of about 100 to 110 amino acids or a larger number of amino acids, and is mainly responsible for antigen recognition. The terms "variable light chain" and "variable heavy chain" mean the variable regions of the light chain and the heavy chain, respectively. In some cases, the antibody, or immune portion of the antibody, can be chemically bound to a fusion protein with another protein or expressed as a fusion protein with another protein.

The CAR of the present invention is expected to bind to protein targets in mammals (particularly humans). The use of protein names may correspond to a mouse or human version of a protein.

The affinity between the binding domain polypeptide and the CAR protein according to the present disclosure can be determined by conventional techniques (eg, competitive ELISA (enzyme-linked immunosorbent assay), binding association, substitution assay with labeled ligand, surface plasmon resonance apparatus (Biacore, etc.), etc. )) Can be easily obtained.

A humanized antibody comprising one or more CDRs of an antibody according to the invention or containing one or more CDRs derived from that antibody can be made using any method known in the art. .. For example, a monoclonal antibody can be humanized using four common steps. The steps are (1) a step of determining the nucleotide sequence of the light chain variable domain and the heavy chain variable domain of the original antibody and the expected amino acid sequence, and (2) a step of designing a humanized antibody, that is, a humanization process. A step of determining which antibody framework region to use, (3) a step of actually using a method / technique for humanization, and (4) a step of transfecting and expressing a humanized antibody. For example, United States Patents No. 4,816,567; No. 5,807,715; No. 5,866,692; No. 6,331,415; No. 5,530,101; No. 5,693,761. See Nos. 5,693,762; Nos. 5,585,089; Nos. 6,180,370; Nos. 5,225,539; Nos. 6,548,640.

The term humanized antibody refers to the fact that at least a portion of the framework region of an immunoglobulin and, in some cases, a CDR region or other region involved in binding is derived from a human immunoglobulin sequence, or human immunity. It means that it is adapted to the globulin sequence. Humanized, chimeric, and partially humanized versions of mouse monoclonal antibodies start with, for example, mouse and / or human genomic DNA sequences encoding H and L chains, or cDNA encoding H and L chains. Starting from a clone, it can be made by recombinant DNA technology (Queen et al., 1989; WO 90/07861). Alternatively, a human monoclonal antibody can be used as the monoclonal antibody used in the method of the present invention. Human antibodies can be obtained, for example, using the phage presentation method (WO 91/17271; WO 92/01047).

As used herein, humanized antibody also means an antibody in non-human (eg, mouse, camel, llama, shark) form, which is a special chimeric immunoglobulin containing the smallest sequence derived from a non-human immunoglobulin, or An immunoglobulin chain, or fragment thereof (eg, Fv, Fab, Fab', F (ab') _2, or other antigen-binding partial sequence of an antibody).

As used herein, human antibodies, humanized antibodies, human antibody fragments, humanized antibody fragments are antibodies having an amino acid sequence corresponding to the amino acid sequence of a human-produced antibody, and / or are known in the art. It means an antibody produced by utilizing any technique for producing a human antibody disclosed in the specification. Human antibodies or fragments thereof can be selected by competitive binding experiments or, otherwise, those having the same epitope specificity as a particular mouse antibody. Surprisingly, the humanized antibodies of the invention share a wide range of useful functional properties of mouse antibodies. Human polyclonal antibodies can also be provided in the form of serum from humans immunized with an immunogen. In some cases, such polyclonal antibodies can be concentrated by affinity purification using amyloid fibrillar and / or non-fibrous polypeptides or fragments thereof as affinity reagents. Monoclonal antibodies can be obtained from serum according to the technique described in WO 99/60846.

Variable region and CDR

The variable region of an antibody, whether alone or in combination, means the variable region of the antibody light chain or the variable region of the antibody heavy chain. The variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs) (also known as hypervariable regions). The CDRs within each strand are held close to each other by FR and combined with the CDRs from the other strand contribute to the formation of the antigen binding site of the antibody.

There are a number of techniques available for determining CDRs, such as an approach based on interspecific variability in sequences (ie Kabat et al., "Sequences of Proteins of Immunological Interest" (5th edition, 1991, National Institutes of Health). , Bethesda, Maryland)); There is an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al. (1997) J. Molec. Biol. Vol. 273: pp. 927-948). Another approach includes the IMGT International ImMunoGeneTics Information System (Marie-Paule Lefranc). The definition of Kabat is based on sequence volatility and is the most commonly used method. The definition of Chothia is based on the location of the loop region of the structure, in which the definition of AbM is from Oxford Molecular's AbM antibody modeling software (www.bioinf.org.uk: Dr. Andrew CR Martin's group). It is a compromise between the two used in. As used herein, CDR means a CDR defined by one or more approaches, or a combination of these approaches.

In some embodiments, the invention provides an antibody or fragment thereof that integrates into CAR, which antibody or fragment thereof is at least one CDR, at least two, at least three, or fragments thereof of an antibody according to the invention. It contains at least one CDR, at least two, at least three, or more CDRs that are substantially the same as more CDRs. Other embodiments are at least two, or substantially the same as at least two, three, or four, five, or six CDRs of an antibody of the invention or derived from an antibody of the invention. It contains antibodies with 3, or 4, or 5, or 6 CDRs. In some embodiments, the at least one, or two, or three, or four, or five, or six CDRs thereof are at least one, two, or three of the antibodies of the invention. At least about 70%, or 75%, or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 95%, or 96%, or 97%, or 98% with CDR. , Or 99% match. For the purposes of the present invention, binding specificity and / or overall activity is generally retained, but the degree of activity can vary (may be greater or smaller) compared to antibodies. Will be understood.

Additional components of CAR

In some embodiments, the CARs considered herein can include linker residues added to allow proper molecular spacing and placement between the various domains. It is, for example, a linker containing an amino acid sequence that connects the VH domain and the VL domain and provides a spacer function that matches the interaction of the two partially bound domains. As a result, the resulting polypeptide retains a specific binding affinity for the same target molecule as the antibody containing the same light chain variable region and heavy chain variable region. The CARs considered herein can include one, two, three, four, five, or more linkers. In a particular embodiment, the length of the linker is about 1 to about 25 amino acids, or about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or amino acids of any length. ..

Examples of linkers include glycine polymers; glycine-serine polymers; glycine-alanine polymers; alanine-serine polymers; other flexible linkers known in the art (such as Whiterow linkers). Since the glycine polymer and the glycine-serine polymer are not comparatively structured, they can function as a neutral tether between the domains of fusion proteins such as CAR described herein.

In a particular embodiment, the binding domain of CAR is followed by one or more "spacers" or "spacer polypeptides". "Spacer" or "spacer polypeptide" means a region that separates the antigen-binding domain from the surface of an effector cell to allow for proper cell / cell contact, antigen binding, and activation. In some embodiments, the spacer domain is the portion of the immunoglobulin containing one or more heavy chain constant regions (eg, CH2 and CH3), but is not limited thereto. The spacer domain can include the amino acid sequence of the native immunoglobulin hinge region or the modified immunoglobulin hinge region. In one embodiment, the spacer domain comprises CH2 and CH3 of IgG1 or IgG4. In one embodiment, mutations in the Fc binding domain of such spacer / hinge regions prevent CAR from binding to Fc receptors expressed on the surface of macrophages and other naturally occurring immune cells. ..

In some embodiments, the binding domain of CAR is followed by one or more "hinge domains". The hinge domain serves to enable proper cell / cell contact, antigen binding, and activation by locating the antigen binding domain away from the surface of the effector cells. The CAR can include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be derived from either a natural source, a synthetic source, a semi-synthetic source, or a recombinant source. The hinge domain can include the amino acid sequence of a native immunoglobulin hinge region or a modified immunoglobulin hinge region. Examples of hinge domains suitable for use in the CARs described herein are derived from the extracellular region of type 1 membrane proteins (CD8α, CD4, CD28, PD1, CD152, CD7, etc.). Yes, this hinge domain may be a wild-type hinge region from these molecules or may be modified. In another embodiment, the hinge domain comprises any of PD1, CD152, CD8α hinge domains.

The "transmembrane domain" is the portion of the CAR that fuses with the extracellular binding portion and the intracellular signaling domain, and anchors the CAR to the plasma membrane of immune effector cells. The TM domain can be derived from either a natural source, a synthetic source, a semi-synthetic source, or a recombinant source. The TM domains are the T cell receptors CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, PD1. It can be derived from α chain, β chain, and ζ chain. In one embodiment, the CARs considered herein include a TM domain derived from CD8α or CD28.

In a particular embodiment, the CARs considered herein include an intracellular signaling domain. The "intracellular signaling domain" is the part of the CAR that is involved in transmitting the message that the anti-CXCR5 CAR successfully bound to the human CXCR5 polypeptide into the immune effector cells, and by that message, the effector. Induced by cell function (eg activation, cytokine production, proliferation, cytotoxic activity (including releasing cytotoxic factors to CAR-bound target cells), and antigen binding to the extracellular CAR domain. Other cellular responses) are induced. The term "effector function" means a specialized function of immune effector cells. For example, as an effector function of T cells, activity including cell lysis activity or assistance, or cytokine secretion is possible. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits effector function signals to cause cells to perform specialized functions. The CARs considered herein include one or more co-stimulation signaling domains for enhancing the effects and / or proliferation and / or memory formation of T cells expressing the CAR receptor. As used herein, "co-stimulating signaling domain" means the intracellular signaling domain of a co-stimulating molecule. Co-stimulatory molecules are cell surface molecules that provide a second signal necessary for the effective activation and function of T lymphocytes when bound to an antigen, other than antigen receptors and Fc receptors.

In one embodiment, the CAR comprises an intracellular domain that includes a co-stimulating domain and a signaling (activating) domain. Thus, the CAR construct comprises an intracellular signaling domain (CD3ζ) of the native T cell receptor complex and one or more co-stimulating domains that provide a second signal that stimulates total activation of T cells. There is. The co-stimulation domain is thought to increase cytokine production in CAR T cells, facilitating T cell replication and T cell persistence. Co-stimulatory domains have also been shown to block the depletion of CAR T cells, increase the antitumor activity of T cells, and enhance the survival of CAR T cells in patients. As a non-limiting example, CAR constructs with a 4-1BB costimulatory domain were found in preclinical studies in T cell subset compositions with progressive sustained proliferation and effector function, increased persistence, and abundance. It was associated with the central memory cell (TCM) that became. 4-1BB is a member of the tumor necrosis factor (TNF) superfamily and is an in vivo inducible glycoprotein receptor that is predominantly expressed on the surface of antigen-activated CD4 and CD8 T cells. As a non-limiting example, CD28 is a member of the immunoglobulin (Ig) superfamily. CD28 is constitutively expressed on the surfaces of resting CD4 T cells and CD8 T cells, and on the surfaces of activated CD4 T cells and CD8 T cells, and T cell activity by stimulating the PI3K-AKT signaling pathway. It plays an extremely important role in the conversion. In one embodiment, the intracellular domain comprises a co-stimulating domain of both 4-1BB and CD28. Another co-stimulation domain includes ICOS and OX40, which can be combined with the CD3ζ signaling (activation) domain.

Polypeptide

"Peptide", "polypeptide", "polypeptide fragment", and "protein" are used interchangeably unless otherwise specified, and are used in the usual sense, that is, in the sense of amino acid sequence. The polypeptide is not limited to a particular length. Polypeptides can include, for example, full-length protein sequences, or fragments of full-length proteins, in addition to post-translational modifications of the polypeptide (eg, glycosylation, acetylation, phosphorylation, etc.), as well as in the art. Other known modifications can also be included. Modifications include both natural and non-natural modifications.

In various embodiments, the CAR polypeptide considered herein comprises a signal (or leader) sequence at the N-terminus of the protein that directs the transfer of the protein at the same time as or after translation. Polypeptides can be prepared utilizing any of a variety of well-known recombinant and / or synthetic techniques. The polypeptides considered herein specifically include the CAR of the present disclosure, or the deletion and / or addition of one or more amino acids as compared to the CAR disclosed herein. , And / or sequences with substitutions.

"Isolated peptides" or "isolated polypeptides" and the like are herein isolated and / or purified in vitro from the cellular environment or from association with other components of the cell. It means a peptide molecule or polypeptide molecule, that is, one that is not significantly associated with an in vivo substance. Similarly, "isolated cell" means a cell obtained from a tissue or organ in vivo and is substantially free from the extracellular matrix.

Nucleic acid

As used herein, the term "polynucleotide" or "nucleic acid molecule" refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), plus-strand RNA (RNA (+)), minus-strand RNA (RNA (-)), It means either genomic DNA (gDNA), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. The polynucleotides of the invention contain at least about 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80% of any reference sequence and sequence described herein. Or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100% matching polynucleotide or variant is preferably included, typically that variant. Maintains the biological activity of at least one of the reference sequences. In the examples of various embodiments, in the present invention, a polynucleotide including an expression vector, a viral vector, a transfer plasmid, a composition, and a cell containing the same are partially considered.

Polynucleotides can be prepared and / or manipulated and / or expressed using any of the well-established and versatile techniques known and available in the art. A nucleotide sequence encoding a polynucleotide can be inserted into a suitable vector to express the desired polypeptide. Examples of vectors are plasmids, autonomously replicating sequences, and transcribed elements. Non-limiting additional examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) PI-derived artificial chromosomes (PAC)), and bacteriophage (λ). Phage, MI 3 phage, etc.), animal virus. Non-limiting examples that fall into the category of animal viruses useful as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (eg herpes simplex virus), natural pox virus, baculovirus, papilloma. A virus, papova virus (eg, SV40). An example of an expression vector is the pClneo vector for expression in mammalian cells (Promega); pLenti4 / V5-DEST ™ for introducing genes mediated by lentivirus and expressing them in mammalian cells. ), PLenti6 / V5-DEST ™, pLenti6.2 / V5-GW / lacZ (Invitrogen). In a particular embodiment, the sequence encoding the chimeric protein disclosed herein can be linked to such an expression vector to express the chimeric protein in mammalian cells. The "regulatory element" or "regulatory sequence" present in the expression vector is the untranslated region of the vector (replication origin, secretory cassette, promoter, enhancer, translation initiation signal (Shine-Dargano sequence or Kozak sequence), intron, Polyadenylated sequences, 5'and 3'untranslated regions (which interact with host cell proteins to perform transcription and translation). Such elements can vary in length and specificity. Any suitable number of transcription and translation elements, including universal promoters and induction promoters, can be used, depending on the vector system and host used.

vector

In a particular embodiment, a retroviral vector (eg, a lentiviral vector) that encodes a CAR on a cell (eg, an immune effector cell such as a T cell). For example, transduction by transduction into immune effector cells with a CAR-encoding vector containing a humanized anti-CXCR5 antibody or antigen-binding fragment that binds to a CXCR5 polypeptide, a transmembrane domain, and an intracellular signaling domain. These cells can induce a CAR-mediated cytotoxic response.

Retroviruses are a common tool for delivering genes. In a particular embodiment, a retrovirus is used to deliver a polynucleotide encoding a chimeric antigen receptor (CAR) to cells. As used herein, the term "retrovirus" means an RNA virus that reverse-transcribes the genomic RNA of the retrovirus into a linear double-stranded DNA copy that then integrates the genomic DNA into the host genome. When a virus is integrated into the host genome, it is called a "provirus." The provirus acts as a template for RNA polymerase II and directs the expression of RNA molecules encoding structural proteins and enzymes required for the production of new viral particles.

Non-limiting examples of retroviruses suitable for use in individual embodiments include Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV). , Mouse breast cancer virus (MuMTV), Tenagazaru leukemia virus (GaLV), Cat leukemia virus (FLV), Spuma virus, Friend mouse leukemia virus, Mouse stem cell virus (MSCV), Rous sarcoma virus (RSV), Lentivirus.

As used herein, the term "wrench virus" means a complex group (genus) of retroviruses. Non-limiting examples of lentiviruses include HIV (human immunodeficiency virus; including HIV1 and HIV2); Bisna-maedivirus (VMV); goat arthritis-encephalitis virus (CAEV); horses. Infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); simian immunodeficiency virus (SIV). In one embodiment, an HIV-based vector skeleton (ie, an HIV cis-acting sequence element) is preferred. In a particular embodiment, a lentivirus is used to deliver a polynucleotide containing CAR to cells.

The term "vector" as used herein means a nucleic acid molecule that is capable of translocating or transporting another nucleic acid molecule. The transferred nucleic acid is generally linked (eg, inserted) to the nucleic acid molecule of the vector. The vector can contain sequences that direct autonomous replication within the cell, or sequences sufficient to allow integration into the DNA of the host cell. Useful vectors include, for example, plasmids (eg, DNA plasmids, RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, viral vectors. Useful viral vectors include, for example, retrievers and lentiviruses that are defective in replication. In yet another embodiment of the invention, a nucleic acid encoding CXCR5 CAR can be inserted via CrispR / Cas and TALEN. Suitable vectors for CRISPR / Cas and TALEN-mediated insertions are known to those of skill in the art.

As will be apparent to those of skill in the art, the term "viral vector" typically refers to a nucleic acid molecule (eg, a transfer plasmid) that contains a virally derived nucleic acid element that facilitates the introduction or integration of the nucleic acid molecule into the genome of a cell. , Or widely used to mean viral particles that mediate the introduction of nucleic acids. Viral particles typically contain a variety of viral components, sometimes in addition to nucleic acids, as well as host cell components.

The term viral vector means a virus or viral particle capable of transferring nucleic acid into a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from the virus. The term "retroviral vector" means a viral vector or plasmid containing structural and functional genetic elements primarily derived from a retrovirus, or a portion thereof.

Therefore, in one preferred embodiment, the invention relates to a method of transfecting a cell with an expression vector encoding CAR. For example, in some embodiments, the vector comprises additional sequences, such as promoters, enhancers, poly-A signals, and / or one or more introns that facilitate the expression of CAR. In a preferred embodiment, the CAR-encoding sequence is flanked by the transposon sequence so that the presence of the transposase can integrate the coding sequence into the genome of the transfected cells.

In some embodiments, the genetically modified cells are further transfected with a transposase that facilitates the integration of the CAR-encoding sequence into the genome of the transfected cells. In some embodiments, the transposase is provided as a DNA expression vector. However, in a preferred embodiment, the transposase is provided as an expressible RNA or protein so that long-term expression of the transposase does not occur in transgenic cells. For example, in some embodiments, the transposase is provided as mRNA (eg, mRNA containing a cap and poly-A tail). Any transposase system can be utilized according to embodiments of the present invention. However, in some embodiments, the transposase is a salmonide-type Tel-like transposase (SB). For example, as a transposase, a so-called "sleeping beauty" transposase is possible. See, for example, US Pat. No. 6,489,458 (the content of which is incorporated herein by reference). In some embodiments, the transposase is an enzyme that has been engineered to increase enzyme activity. Some non-limiting examples of transposases include SB 10, SB 11, SB 100X transposases (eg, Mates et al., 2009, Nat Genet. Vol. 41 (6): 753-761). , Or US Pat. No. 9,228,180 (these are incorporated herein by reference). For example, one method can include electroporating cells with mRNA encoding any of SB 10, SB 11, or SB 100X transposase.

Sequence variant:

Sequence variants of nucleic acids, proteins, antibodies, antibody fragments, CARs (eg, those defined by% sequence matching) that maintain binding properties similar to those of the present invention are also included within the scope of the invention. Such variants that differ from the specific sequences presented but retain substantially the same binding properties (such as target specificity) are functional analogs or functionally similar. Known as. Sequence matching is related to the percentage of nucleotides or amino acids that match when performing sequence alignment.

As used herein, the reference to "sequence match" means to some extent that the sequence is the same per nucleotide or amino acid on the comparison window. Therefore, the "sequence match rate" compares two optimally aligned sequences on the comparison window and has the same nucleobase (eg A, T, C, G, I) or the same amino acid residue (eg A, T, C, G, I) in both sequences For example, Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Ph, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, Met) are found and matched. The number of matched positions is calculated, the number of matched positions is divided by the total number of positions in the comparison window (that is, the size of the window), and the result is multiplied by 100 to obtain the rate of sequence matching. Included are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% sequence match with any reference sequence described herein. , 96%, 97%, 98%, 99%, 100% of nucleotides and polypeptides, where typically the polypeptide variant exhibits at least one biological activity of the reference polypeptide. Maintained.

As will be appreciated by those skilled in the art, there are numerous nucleotide sequences encoding the polypeptides described herein as a result of the degeneracy of the genetic code. Some of these polynucleotides have minimal homology or sequence matching with the nucleotide sequence of any native gene. Nevertheless, different polynucleotides due to differences in codon utilization are specifically considered in the present invention. Deletions, substitutions, and other changes in sequences that apply to sequence matching are also included in the invention.

Modifications of protein sequences that may occur through substitutions are also included within the scope of the invention. The substitutions defined herein are modifications made to the amino acid sequence of a protein, in which one or more amino acids are replaced with the same number of (different) amino acids, which is different from the original protein. A protein containing the sequence is produced. Substitutions can be performed that preferably do not significantly alter the function of the protein. As with addition, natural and artificial substitutions are possible as substitutions. It is well known in the art that amino acid substitutions can be performed without significantly altering protein function. This is especially true if the modification involves a "conservative" amino acid substitution that replaces one amino acid with another amino acid with similar properties. Such "conserved" amino acids can be natural or synthetic amino acids that can be replaced without significantly affecting the structure and function of the protein because of size, charge, polarity and conformation. .. Many amino acids are often replaced with conservative amino acids without adversely affecting protein function.

In general, the non-polar amino acids Gly, Ala, Val, Ile, Leu; the non-polar aromatic amino acids Ph, Trp, Tyr; the neutral polar amino acids Ser, Thr, Cys, Gln, Asn, Met; the positively charged amino acids Lys, Arg, His; Negatively charged amino acids Asp, Glu are representative groups of conserved amino acids. This list is not exhaustive. For example, Ala, Gly, Ser and sometimes Cys belong to different groups but can replace each other.

In the substitution variant, at least one amino acid residue in the antibody molecule has been removed and a different residue has been inserted in its place. The most interesting sites for substitution mutagenesis include hypervariable regions, but changes in FR are also considered. If the biological activity changes as a result of such substitutions, as labeled "representative substitutions" in the table immediately below, or further described below with respect to the class of amino acids. As such, more changes can be introduced to screen the product.

Substantial alterations in the biological properties of an antibody include (a) maintenance of the structure of the polypeptide backbone (eg, sheet or helical structure) in the region of substitution, or (b) maintenance of the charge or hydrophobicity of the molecule at the target site. , Or (c) by selecting substitutions that have a significantly different effect on maintaining side chain size.

Conservative amino acid substitutions are not limited to natural amino acids, but also include synthetic amino acids. Commonly used synthetic amino acids are neutral non-polar analogs, ω amino acids with varying chain lengths, cyclohexylalanine; neutral non-polar analogs citrulline and methionine sulfoxide, neutral aroma. Phenylglycine, a group analog; citrulline, a negatively charged analog, and ornithine, a positively charged amino acid analog. Like natural amino acids, this list is not exhaustive and is just an example of well-known substitutions in the art.

Genetically modified cells and immune cells

In a particular embodiment, cells genetically modified to express the CARs considered herein are considered for use in the treatment of B cell-related diseases. As used herein, the expression "genetically engineered" or "genetically modified" means adding additional genetic material in the form of DNA or RNA to the entire intracellular genetic material. The terms "genetically modified cells," "modified cells," and "redirected cells" are used interchangeably. As used herein, the term "gene therapy" refers to the permanent or temporary introduction of additional genetic material in the form of DNA or RNA into all genetic material in a cell to repair, modify, or modify a gene. , Means expressing a therapeutic polypeptide (eg, CAR). In a particular embodiment, the CARs considered herein are introduced into immune effector cells and expressed, redirecting their specificity to a target antigen of interest (eg, a CXCR5 polypeptide).

An "immune cell" or "immune effector cell" is any cell of the immune system that has one or more effector functions (eg, killing activity of cytotoxic cells, secretion of cytokines, induction of ADCC and / or CDC). Immune effector cells can also be differentiated from iPSCs (induced pluripotent stem cells).

The immune effector cells of the present invention can be autologous / self-generated (“self”) cells or non-autologous (“non-self”, eg, allogeneic, or allogeneic, or heterologous) cells. "Self" is, herein, meant to be derived from the same subject and is a preferred embodiment of the invention. By "self-generated" is meant herein that they are derived from the same species and are genetically different from the cells being compared. By "synonymous" is used herein to mean that they are derived from different subjects and are genetically identical to the cells being compared. By "heterogeneous" is meant herein that it is derived from a different species than the cells being compared. In a preferred embodiment, the cells of the invention are autologous cells or self-generated cells.

Examples of immune effector cells used in CAR considered in the present invention include T lymphocytes. The term "T cell" or "T lymphocyte" is recognized in the art and includes thymocytes, immature T lymphocytes, mature T lymphocytes, quiescent T lymphocytes, and cytokine-induced killer. Cells (CIK cells), activated T lymphocytes. Cytokine-induced killer (CIK) cells are typically CD3-positive and CD56-positive natural killer (NK) -like T lymphocytes with no major histocompatibility complex (MHC) restriction. As T cells, T helper (Th; CD4 ⁺ T cell) cells, for example, T helper 1 (Th1) cells or T helper 2 (Th2) cells can be used. The T cells can be cytotoxic T cells (CTL; CD8 ⁺ T cells), CD4 ⁺ CD8 ⁺ T cells, CD4CD8 T cells, or any other subset of T cells. Examples of other populations of T cells suitable for use in special embodiments include naive T cells, memory T cells, stem cell-like memory cells (TSCM).

For example, the CAR-modified T cells of the invention as described herein can recognize and kill tumor cells when reintroduced into a patient after transplantation of autologous cells. CIK cells can have greater cytotoxic activity than other T cells, which is a preferred embodiment of the immune cells of the present invention.

As will be appreciated by those skilled in the art, other cells can also be used as immune effector cells with the CARs described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include progenitor cells of effector cells, which can be induced to differentiate such progenitor cells in vivo or in vitro to become immune effector cells. As a progenitor cell, iPSC that becomes an immune effector cell under predetermined culture conditions is possible.

INDUSTRIAL APPLICABILITY The present invention provides a method for producing an immune effector cell expressing CAR considered herein. In one embodiment, the method comprises expressing one or more CARs described herein in the immune effector cells by transducing or transducing the immune effector cells isolated from the individual. I'm out. In some embodiments, immune effector cells are isolated from an individual and genetically modified, but are not further manipulated in vitro. Such cells are then re-administered directly to the individual. In yet another embodiment, immune effector cells are first activated, stimulated to proliferate in vitro, and then the gene is modified to express CAR. In this regard, immune effector cells can be cultured prior to and / or after genetic modification (ie, transducing or transfecting to express the CARs considered herein).

In a particular embodiment, the source of the cells is obtained from the subject prior to in vitro manipulation or genetic modification of the immune effector cells described herein. In a particular embodiment, the CAR-modified immune effector cells contain T cells. T cells can be obtained from multiple sources. Non-limiting examples of sources include peripheral blood monocyte cells, bone marrow, lymph node tissue, cord blood, thymic tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, T cells are a technique of any of a number of techniques known to those of skill in the art (precipitation (eg, Ficoll ™ separation), antibody-bound bead-based method (MACS ™). ) Separation (Miltenyi))) can be used to obtain from 1 unit of blood collected from the subject. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. Aferesis products typically include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells recovered by apheresis can be washed to remove the plasma fraction and the cells can be placed in a suitable buffer or medium for subsequent treatment. Cells can be washed with PBS or with any other suitable solution that is calcium and magnesium free and also free of most, if not all, divalent cations. As will be appreciated by those skilled in the art, the cleaning process can be accomplished by methods known to those skilled in the art (eg, semi-automated flow-through centrifugation). For example, Cobe 2991 cell processor, Baxter CytoMate, etc. After washing the cells, they can be resuspended in various biocompatible buffers and other saline solutions with or without buffers. In some embodiments, the unwanted constituents of the apheresis sample can be removed in the medium in which the cells are directly resuspended.

In some embodiments, isolation of T cells from peripheral blood monocytic cells (PBMCs) is achieved by lysing erythrocyte cells and depleting monocytes, for example by centrifugation through a PERCOLL ™ gradient. .. A particular subpopulation of T cells can be further isolated by a positive selection technique or a negative selection technique. One method utilized herein is cell sorting and / or selection through negative magnetic immunoadsorption or flow cytometry with a cocktail of monoclonal antibodies directed at cell surface markers present on the surface of negatively selected cells. ..

The methods considered herein can be used to directly genetically modify PBMCs to express CAR. In some embodiments, after isolation of PBMCs, T lymphocytes are further isolated, and in some embodiments, cytotoxic T lymphocytes and helper T lymphocytes before or after gene modification and / or proliferation. Both lymphocytes can be classified into naive T cell subpopulations, memory T cell subpopulations, and effector T cell subpopulations. CD8 ⁺ cells can be obtained using standard methods. In some embodiments, the operation of further classifying CD8 ⁺ cells into naive central memory cells and effector cells is performed by identifying cell surface antigens associated with each of these types of CD8 ⁺ cells.

In some embodiments, the immune cells of the invention (eg, T cells described herein) can be obtained from induced pluripotent stem cells (iPSCs) using methods known to those of skill in the art. ..

Acceptable approaches to generate CAR T cells rely on genetic modification and proliferation of circulating mature T cells. Because these methods utilize autologous T cells, they reduce the risk of graft-versus-host (GvHD) disease from allogeneic T cells through the expression of endogenous TCRs and reject through MHC incompatibility. Decreases. As an alternative, genetic modification to express the CAR of the invention by directly differentiating engineered T cells from pluripotent stem cells (such as inducible pluripotent stem cells) in vitro. A virtually unlimited source of cells is provided. In some embodiments, the so-called master iPSC system, which is an updatable source for the steady and iterative production of homogeneous cell products, can be maintained. In some embodiments, transforming the master iPSC cell line with a nucleic acid encoding CAR is considered prior to proliferation and differentiation into the desired immune cells (preferably T cells). Since T lymphocytes can be generated from, for example, iPSCs, it is considered that iPSCs can be modified with a nucleic acid encoding CAR, then proliferated for administration to patients and differentiated into T cells. It would also be possible to differentiate the iPSC into suitable immune cells (such as T cells), then transform with a nucleic acid encoding CAR, proliferate and administer. In order to provide a suitable number of cells for administration, any possible combination of iPSC proliferation, genetic modification, and proliferation is considered in the present invention.

After isolation using known methods, the genes of immune effector cells (such as T cells) were modified, or immune effector cells were activated and proliferated in vitro (differentiated in the case of progenitor cells). ) Later, the gene can be modified. In one particular embodiment, the genes of immune effector cells (such as T cells) have been modified (eg, transduced with a viral vector containing a nucleic acid encoding CAR) with the chimeric antigen receptor considered herein and then in vitro. Activated and propagated in. In various embodiments, T cells can be activated and proliferated to express CAR before or after genetic modification. The method to be used at that time is described, for example, US Pat. No. 6,352,694; No. 6,534,055; No. 6,905,680; No. 6,692,964; No. 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,7, No. 232,566; No. 7,175,843; No. 5,883,223; No. 6,905,874; No. 6,797,514; No. 6,867,041; United States Patent Application Publication No. It is 2006-0121005.

In yet another embodiment, a mixture of vectors (eg, one, two, three, four, five, or more vectors) is used in modifying the genes of immune effector cells in the donor population. Can be used. In this case, each vector encodes a different chimeric antigen receptor protein considered herein. The resulting modified immune effector cells form a mixed population of modified cells and contain a proportion of the modified cells expressing two or more different CAR proteins.

In one embodiment, according to the present invention, cells are alive upon thawing as a method of storing genetically modified immune effector cells expressing a mouse, human, or humanized CAR protein and targeting the CXCR5 protein. Methods are provided that involve keeping the immune effector cells cold to remain intact. A permanent source of such cells for future treatment of patients suffering from B cell-related diseases by keeping some of the immune effector cells expressing the CAR protein at low temperatures by methods known in the art. Can be provided. When needed, the transformed immune effector cells kept at low temperature are thawed, grown and proliferated to increase the number of such cells.

Compositions and formulations

The compositions considered herein can include one or more polypeptides, polynucleotides, vectors containing polynucleotides, genetically modified immune effector cells, etc. considered herein. Non-limiting examples of compositions include pharmaceutical compositions. A "pharmaceutical composition" is a composition in the form of a pharmaceutically acceptable or physiologically acceptable solution for application to cells or animals alone or in combination with one or more other therapeutic modalities. Means things. If desired, the compositions of the invention can be combined with other agents (eg, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, and various other pharmaceutically active agents). It will also be understood that they can be administered in combination. There are substantially no restrictions on the other components that can be included in the composition, provided that the additional agent does not adversely affect the efficacy of the composition to provide the intended treatment.

The expression "pharmaceutically acceptable" is used herein as a reasonable benefit / without excessive toxicity, irritation, allergic reactions, no other problems or complications, within the scope of sound medical judgment. At risk ratio, it means a compound and / or material, and / or composition, and / or dosage form suitable for use in contact with human and animal tissues.

As used herein, non-limiting examples of "pharmaceutically acceptable bases or diluents, or excipients" are included by the United States Food and Drug Administration as acceptable for use in humans and livestock. Any approved adjuvants, bases, excipients, flow promoters, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents , Stabilizers, isotonic agents, solvents, surfactants, emulsifiers. Non-limiting examples of pharmaceutically acceptable bases include sugars (lactos, glucose, sucrose, etc.); starch (corn starch, potato starch, etc.); cellulose and its derivatives (sodium carboxymethyl cellulose, ethyl cellulose, etc.). Cellulose acetate, etc.); Tragacanto gum; Maltese; Gelatin; Tarku; Cacao butter, wax, animal and plant fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils (peanut oil, cottonseed oil, benibana oil, sesame oil, olive oil, Corn oil, soybean oil, etc.); Glycol (propylene glycol, etc.); Polyol (glycerin, sorbitol, mannitol, polyethylene glycol, etc.); Ester (ethyl oleate, ethyl laurate, etc.); Agar; Buffer (magnesium hydroxide, water) (Aluminum oxide, etc.); Arginic acid; Exothermic substance-removed water; Isotonic physiological saline; Ringer's solution; Ethyl alcohol; Phosphate buffer solution; Any other compatible substance used in pharmaceutical formulations.

In a particular embodiment, the composition of the invention comprises an amount of CAR-expressing immune effector cells considered herein. As used herein, the term "amount" refers to the "effective amount" of genetically modified therapeutic cells (eg, T cells) to achieve effective or desired prophylactic or therapeutic outcomes (including clinical outcomes). Or it means "effective amount".

"Prophylactically effective amount" means the amount of genetically modified therapeutic cells effective in achieving the desired prophylactic result. Because prophylactic doses are used in subjects prior to the early stages of the disease, or in subjects in the early stages of the disease, prophylactic doses are typically less than therapeutically effective, but not necessarily. Not exclusively. The term prevention does not necessarily mean complete suppression or prevention of a particular medical disorder. The term prevention also means a reduced risk of developing a medical disorder or exacerbating the symptoms of that medical disorder.

The "therapeutic amount" of genetically modified therapeutic cells can vary depending on a variety of factors, including the individual's disease state, age, gender, body weight, and the ability of stem and progenitor cells to elicit the desired response to the individual. There is sex. A therapeutically effective amount also means an amount in which the beneficial effects of the treatment outweigh any toxic or detrimental effects of the viral or transduced therapeutic cells. The expression "therapeutically effective amount" includes an amount effective to "treat" a subject (eg, a patient). When the amount for treatment is indicated, the exact amount of the composition of the invention to be administered will be determined by the physician regarding age, body weight, tumor size, degree of infection or metastasis, condition of the patient (subject). It can be decided in consideration of individual differences.

As a general rule, the T cell-containing pharmaceutical compositions described herein are administered at a dose of 10 ² to 10 ¹⁰ cells per kg body weight, preferably 10 ⁵ to 10 ⁷ cells per kg body weight. The dose can include any integer value within this range. The number of cells will depend on the end use of the composition and the type of cells contained therein. For the uses shown herein, cells are generally no more than 1 liter in volume and can be no more than 500 ml, or even 250 ml or 100 ml or less. The density of thus desired cell is typically 10 ⁶ cells / ml, greater than typically 10 ⁷ cells / ml, or greater than typically 10 ⁸ cells / ml, or greater than a density greater than that. It can be divided clinically significant numbers of immune cells into a plurality of infusion and accumulate it, 10 ⁵ cells or more, ¹⁰⁶ cells or more, 10 ⁷ cells or more, 10 ⁸ It can be any of 10 cells or more, 10 ⁹ cells or more, 10 ¹⁰ cells or more, 10 ¹¹ cells or more, and 10 ¹² cells or more. In some aspects of the invention, fewer cells can be administered, especially because all cells infused are redirected to a particular target antigen. Cell compositions expressing CAR can be administered multiple times at doses within these ranges. The cells can be allogeneic, allogeneic, heterologous, or autologous to the patient being treated.

In general, compositions containing cells that have been activated and proliferated as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. In particular, compositions containing CAR-modified T cells of the invention can be administered alone or as a base and / or diluent, and / or excipient, and / or other constituents (IL-2 and the like). It can be administered as a pharmaceutical composition in combination with a cytokine other than the above, a cell population). In a particular embodiment, the pharmaceutical composition considered herein is an amount of genetically modified T cells that is pharmaceutically or physiologically acceptable for one or more bases, diluents, or excipients. Included in combination with.

The pharmaceutical compositions of the present invention comprising a population of immune effector cells expressing CAR (such as T cells) include buffers (neutral buffered physiological saline, phosphate buffered physiological saline, etc.); carbohydrates (glucose, mannitol, etc.). , Sucrose, dextran, mannitol, etc.); proteins; polypeptides or amino acids (such as glycine); antioxidants; chelating agents (EDTA, glutathione, etc.); adjuvants (eg, aluminum hydroxide); preservatives. The pharmaceutical composition of the present invention is preferably prepared for parenteral administration (for example, intravascular administration (intravenous administration or intraarterial administration), intraperitoneal administration, intramuscular administration).

The liquid pharmaceutical composition, whether in solution, suspension or other form, can contain a sterilizing diluent (water for injection, physiological saline solution (preferably physiological saline solution)). , Ringer solution, isotonic sodium chloride, etc.), non-volatile oil (such as synthetic monoglyceride or diglyceride, which can function as a solvent or suspension medium), polyethylene glycol, glycerin, propylene glycol, other solvents) Antibacterial agents (benzyl alcohol, methylparaben, etc.); Antioxidants (ascorbic acid, sodium hydrogen sulfite, etc.); Chelating agents (ethylenediamine tetraacetic acid, etc.); Buffers (acetate, citrate, phosphate, etc.); Tension One or more of the agents for regulating (sodium chloride, dextrose, etc.). Parenteral preparations can be encapsulated in glass or plastic ampoules, disposable syringes, and multi-dose vials. The injectable pharmaceutical composition is preferably sterilized.

In one particular embodiment, the compositions considered herein comprise an effective amount of immune effector cells expressing CAR alone or in combination with one or more therapeutic agents. Therefore, CAR-expressing immunoeffector cell compositions are applied alone or in combination with other known cancer therapies (radiotherapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc.). be able to. The composition can also be administered in combination with an antibiotic. Such therapeutic agents are acceptable in the art as standard treatments for certain disease states (such as certain cancers) described herein. Typical therapeutic agents to consider include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, and other activators and auxiliaries.

Treatment

The genetically modified immunoeffector cells discussed herein are those of medical disorders associated with the presence of pathogenic cells expressing CXCR5, including non-limiting examples of immunomodulatory diseases and hematological malignancies. Provide improved adoptive immunotherapy for therapeutic use.

In the present specification, "medical disorder related to the presence of pathogenic cells expressing CXCR5" means a medical disease such as cancer or an autoimmune disease, and the cells involved in the pathophysiology of the disease are It expresses CXCR5. Expression of CXCR5 can be demonstrated by a variety of methods known to those of skill in the art, eg, cells are isolated from patients, the cells are evaluated by PCR using primers for the CXCR5 transcript and with anti-CXCR5 antibody. It can be clarified by immunostaining or analysis by flow cytometry. Such pathogenic cells are typically pathogenic mature B cells and / or memory B cells, and / or pathogenic T cells and / or T follicular helper cells.

In a particular embodiment, the composition comprising CAR-modified T cells considered herein is used for the treatment of hematological malignancies. Non-limiting examples of hematological malignancies include B cell malignancies such as non-Hodgkin's lymphoma (NHL) (B cell NHL, T-cell non-Hodgkin's lymphoma with or without leukemic tumor cell dissemination, etc.). ..

Non-Hodgkin's lymphoma includes a large group of cancers of lymphocytes (white blood cells). Non-Hodgkin's lymphoma can develop at any age and is often characterized by larger than normal lymph nodes, fever, and weight loss. Non-Hodgkin's lymphoma may also be present in the extranodes (central nervous system, mucosal tissue (lungs, small intestine, large intestine, gastrointestinal tract)). There are many different types of non-Hodgkin's lymphoma. For example, non-Hodgkin's lymphoma can be divided into a medium-grade (fast-growing) type and a low-grade (slow-growing) type.

Non-Hodgkin's lymphoma can be derived from B cells and T cells. As used herein, the term "non-Hodgkin's lymphoma" includes both "B-cell" non-Hodgkin's lymphoma and "T-cell" non-Hodgkin's lymphoma. B-cell non-Hodgkin's lymphoma (NHL) includes Berkit's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, and immunoblasts. Large cell lymphoma, precursor B lymphoblastic lymphoma, and mantle cell lymphoma. Lymphoma that develops after transplanting bone marrow or stem cells is usually B-cell non-Hodgkin's lymphoma.

T-cell lymphoma accounts for about 15% of all NHL in the United States. There are many different forms of T-cell lymphoma, including angioimmunoblastic T-cell lymphoma (AITL), which is a mature T-cell lymphoma of blood or lymphatic immunoblasts. Yet another form of T-cell lymphoma is associated with cutaneous T-cell lymphoma and T-cell lymphoma with leukemic dissemination.

Chronic lymphocytic leukemia (CLL) can also be treated with the CAR of the present invention. CLL is a low-grade (slow-growth) cancer that causes a slow increase in immature white blood cells (B lymphocytes). Cancer cells spread through the blood and bone marrow and can also affect lymph nodes and other organs (liver, spleen, etc.). CLL eventually causes the bone marrow to fail. Another name for this disease is small lymphocytic lymphoma, which is usually localized to secondary lymphoid organs (eg, lymph nodes, spleen).

In one embodiment of the invention, CAR, or immune cells expressing CAR, are used in the treatment of autoimmune diseases, preferably in the treatment of autoantibody-dependent autoimmune diseases, preferably in the treatment of autoimmune diseases having inflammatory components. It is supposed to be used.

Dysregulation of follicular T-helper (Tfh) cells expressing CXCR5 is an important mechanism that contributes to the abnormal humoral immune response and autoantibody production in autoimmune diseases. Therefore, Tfh cells expressing CXCR5 are practical targets in the context of autoimmunity.

The choice of autoimmune diseases to treat is Takayasu's arteritis, giant cell arteritis, familial Mediterranean fever, Kawasaki disease, nodular polyarteritis, cutaneous nodular polyarteritis, hepatitis-related arteritis, Bechet's disease, Wegener. Granulomatosis, ANCA vasculitis, Charg-Strauss syndrome, microscopic polyangiitis, vasculitis of connective tissue disease, Henoch-Schoenlein purpura, cryoglobulin vasculitis, cutaneous leukocyte-destroying vasculitis, tropical aortitis, sarcoidosis , Kogan Syndrome, Wiscott-Aldrich Syndrome, Leprosy Arthritis, CNS Localized Angiitis, Obstructive Thrombotic Angiitis, Tumor Concomitant Artitis, Uryalysis, Degos's Disease, Myelodystrophy Syndrome, Enduring Elevated Red Spot , Takayasu's globulin D, allergic rhinitis, bronchial asthma, chronic obstructive pulmonary disease, periodontitis, rheumatoid arthritis, atherosclerosis, amyloidosis, Crohn's disease, ulcerative colitis, autoimmune myitis, diabetes, Gillan- Valley syndrome, histiocytosis, osteoarthritis, atopic dermatitis, periodontitis, chronic sinusitis, psoriasis, psoriatic arthritis, microcolonitis, pulmonary fibrosis, glomerulonephritis, whipple disease, still disease , Nodular erythema, otitis media, cryoglobulinemia, Schegren's syndrome, erythematosus (preferably systemic erythematosus (SLE)), regenerative anemia, myelopathy, chronic inflammatory demyelinating polyneuritis, Kimura's disease Takayasu's arteritis, chronic periarteritis, chronic prostatic inflammation, idiopathic pulmonary fibrosis, chronic granulomatosis, idiopathic acarasia, bleomycin-induced pulmonary inflammation, citarabin-induced pulmonary inflammation, autoimmune thrombocytopenia, autoimmune favor Splasia, autoimmune hemolytic anemia, autoimmune lymphopenia, Shagas disease, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto thyroiditis, atrophic thyroiditis, Basedou disease, polyglandular Autoimmune Syndrome, Autoimmune Azison's Disease, Blast Vulgaris, Decolial Blast, Herpes Dermatitis, Autoimmune Alopecia, White spots, Antiphospholipid antibody syndrome, Severe myasthenia, Stiffman syndrome, Good It is most preferably made from pasture syndrome, sympathetic ophthalmitis, folliculitis, sharp syndrome, Evans syndrome, especially from pollinosis, periodontitis, atherosclerosis, rheumatoid arthritis. SLE is preferred.

Systemic lupus erythematosus (SLE) (also known as lupus) is an autoimmune disease in which the body's immune system attacks healthy tissues in different parts of the body. Symptoms vary from person to person and can range from mild to severe. Common symptoms include painful and swollen joints, fever, chest pain, hair loss, mouth ulcers, swollen lymph nodes, tiredness, and a rash (most often on the face).

Follicular T-helper (Tfh) cells were recently discovered to be CD4 ⁺ T-helper cells, which are the major cell reservoir for the human immunodeficiency virus (HIV) (Leon et al., 2017, Frontiers in Immunology, Therefore, CAR immune cells expressing the CAR of the present invention may be able to target the follicular reservoir of HIV-producing T-helper cells (Volume 8: page 622). Therefore, in the treatment of HIV, the CAR of the present invention can be used to remove or suppress the reservoir of HIV-producing T-helper cells. Thus, in one embodiment, the reservoir of HIV-producing T helper cells is considered to be a group of pathogenic cells expressing CXCR5, particularly pathogenic T cells.

In this specification, the terms "individual" and "subject" are often used interchangeably, making use of gene therapy vectors, cell-based therapeutic agents, and methods disclosed elsewhere herein. Means any animal that exhibits a treatable disease, disorder, or symptom of the disease. In a preferred embodiment, the subject is any animal exhibiting symptoms of a hematopoietic disease, injury, disease (eg, a B cell malignancy), a cell-based therapeutic agent disclosed herein. And can be treated by the method. Suitable subjects include laboratory animals (mice, rats, rabbits, guinea pigs, etc.), domestic animals, domesticated animals or pets (cats, dogs, etc.). Subjects include non-human primates and human patients, preferably human patients. Typical subjects include human patients with B cell malignancies, human patients diagnosed with B cell malignancies, or human patients at risk for B cell malignancies.

As used herein, "treating" or "treating" includes any effective or desirable effect on the symptoms or pathology of a disease or pathological condition, including the disease or disease being treated. A negligible reduction in one or more measurable markers of the condition can be included. Treatment can optionally include alleviation or amelioration of symptoms of the disease or pathological condition, as well as delayed progression of the disease or pathological condition. "Treatment" does not necessarily include eradication or complete cure of the disease or pathological condition and its associated symptoms.

As used herein, the terms "prevent" and similar terms such as "prevented," "preventing," and "preventive" prevent the development or recurrence of a disease or pathological condition. , Or an approach that suppresses or reduces. These terms also mean delaying the onset or recurrence of a disease or pathological condition, or delaying the onset or recurrence of symptoms of a disease or pathological condition. As used herein, the term "prevention" and similar terms refer to the degree and / or effect, and / or symptom, and / or load of the disease or condition before the onset or recurrence of the disease or condition. It also includes mitigation of.

In one embodiment, a method of treating a B cell-related pathological condition in a subject in need of treatment comprises an effective amount of a composition comprising the genetically modified immune effector cells considered herein (eg, therapeutically effective). Amount) includes administration. The amount and frequency of administration depends on factors such as the patient's condition, the type and severity of the patient's disease, but the appropriate dose can be determined by clinical trials.

Administration of the compositions considered herein can be performed in any convenient manner, including aerosol inhalation, injection, ingestion, infusion, implantation, and transplantation. In one preferred embodiment, the composition is administered parenterally. The terms "parenteral administration" and "administered parenterally" are used herein to mean administration forms other than enteral and topical administration, usually by injection, without limitation. Examples include intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, enteral, subcutaneous, subepithelial, and intra-articular. , Subcapsular, subcapsular, intraspinal, intrathoracic injections and infusions. In one embodiment, the compositions considered herein are administered to a subject by direct injection into a tumor, lymph node, site of infection.

Drawing

FIG. 1: Schematic diagram of a preferred CAR structure. The VL and VH domains of the antigen binding domain are shown including the linker located between those VL and VH domains. Spacer regions located between the antigen binding domain and the transmembrane domain are also shown. Variants of the intracellular domain have also been shown, which include, for example, the costimulatory domain and the activated domain.

FIG. 2: Schematic diagram of preferred CAR constructs H28, R28, HBB1, HBB2, H28BB. Preferred constructs of the present invention include variants of transmembrane domains, costimulatory domains, and activation domains. In essence, preferred embodiments of the invention allow for the exchange of these various domains, preferably the various domains of the special embodiments disclosed herein, It also includes additional domains with similar functionality known to those skilled in the art.

FIG. 3: A list of possible combinations of the preferred CAR constructs described herein and the various structural elements of CAR.

FIG. 4: Sequence comparison between the mAb binding region and the preferred humanized sequence used in the CAR of the present invention. Alignments are displayed for examining sequence matching between rat hHC and humanized hHC sequences, and between rat hLC and humanized hLC sequences. As can be seen from this indication, the sequence match between the humanized hHC sequence and the rat hHC sequence is clear to be 89%, and the sequence match between the humanized hLC sequence and the rat hLC sequence is 93%. Is clear.

FIG. 5: A sequence diagram of the DNA sequence encoding the mAb binding region hHC, particularly showing a sequence comparison of the original DNA sequence encoding humanized hHC and the codon-optimized (CO) DNA sequence.

FIG. 6: Sequence of the DNA sequence encoding the mAb binding region hLC, particularly showing a sequence comparison of the original DNA sequence encoding humanized hLC and the codon-optimized (CO) DNA sequence.

FIG. 7: GeneArt ™ plasmid with humanized CXCR5-CAR sequence and GeneArt ™ plasmid with rat CXCR5-CAR sequence. The resected scFv was demonstrated using gel electrophoresis.

Figure 8: Gel electrophoresis of the construct and vector after restriction. The plasmid MP71 containing the construct encoding CAR is also displayed.

FIG. 9: Confirmation by flow cytometry that CXCR5-CAR is expressed on the surface of human T cells after transduction using a retrovirus: expression of CAR, construct H28, construct SP6, and no transduction.

FIG. 10: (A) Expression of CXCR5 on the surface of several types of cells evaluated in the functional assay. We evaluated DOHH-2, SU-DHL4, OCI-Ly7, JeKo-1 which are B-NHL cell lines, MCL xenografts derived from primary patients, and NALM6 which is a B-ALL cell line. They were REH, NCI-H929, which is an MM cell line, and Jurkat, which is a T-ALL cell line. As can be seen from the analysis, CXCR5 was expressed on the surface of the B-NHL DOHH-2 cell line, SU-DHL4 cell line, OCI-Ly7 cell line, and JeKo-1 cell line. (B) To rule out that the CXCR5 CAR-T cells of the present invention cross-reactive with healthy human tissue, the expression of CXCR5 was evaluated in a population of primary cells derived from healthy human tissue. Any primary human cell examined (HUVEC, human umbilical vein endothelial cell; HUAEC, human umbilical artery endothelial cell; HA, human astrocyte; HN, human neuron; HPNC, human neuroperitoneal cell; HCoEpiC, human colon epithelial cell) Anti-CXCR5 immunostaining and flow cytometric analysis showed no expression of CXCR5 on the surface. (C) Selected B-NHL cell lines (SU-DHL4, OCI-Ly7, DOHH-2, SC-1, JeKo-1, MEC-1, JVM-3), MM cell lines (NCI-H929), B-ALL cell line (REH, NALM-6), T-All cell line (Jurkat), colon adenocarcinoma cell line (SW-620), untransfected embryonic renal cell line (HEK293), CXCR5 transfected Quantitative measurements of CXCR5 density per cell on the surface of the embryonic renal cell line (HEK-CXCR5) were performed using QuantiBRITE PE calibration beads and CXCR5-specific antibodies.

FIG. 11: Co-culture of CAR transduced human T cells with various target cell lines reveals specific T cell activation by distinguishable CXCR5 ⁺ B-NHL and control cell lines. Functional in vitro co-culture and IFN-γ ELISA were performed. The level of IFN-γ released indicates activation of T cells. No transduction (UT; left bar of each series), T cells expressing CXCR5-CAR (CXCR5 (H28); center bar of each series), SP6 T cells (SP6; right bar of each series) ) Was co-cultured with the target cell lines DOHH-2, SU-DHL4, OCI-Ly7, JeKo-1, NALM6, REH, NCI-H929, and Jurkat. Target cells DOHH-2, SU-DHL4, OCI-Ly7, JeKo-1 exhibit specific release of IFN-γ in response to treatment with the CXCR5 CAR-T cells of the invention. The JVM-3 also shows the release of IFN-γ after treatment.

Figure 12: Cytotoxicity assay reveals selective killing of CXCR5-positive cell lines. Substantially no killing was seen in the CXCR5-negative cell line. Two independent functional in vitro culture assays and a ⁵¹ Cr release assay were performed. (A, B) Target cell lines DOHH-2, SU-DHL4, OCI-Ly7, SC-1 ((B) only), JeKo-1 show lysis after treatment with CAR-T of the present invention. However, cells not expressing CXCR5 (Nalm6 and NCI-H929) do not show lysis.

FIG. 13: CXCR5-redirected CAR-T cells are effective against B-cell non-Hodgkin's lymphoma (B-NHL) in a xenografted NSG mouse model. To prove the idea that the strong in vitro activity of T cells modified with CXCR5 CAR leads to effective antitumor activity in vivo, NOD. In a cohort of Cg-Prkdc ^scid Il2rg ^{tm1 Wjl} / SzJ (NSG) mice, 5 × 10 ⁵ mantle cell lymphoma cells (MCL) JeKo-1 were intravenously inoculated (FIGS. 13A-C) in tandem with GFP. The luciferase gene was transduced. Since NSG mice do not develop T cells, B cells, and NK cells, they are suitable for tolerance and proliferation of xenografted human cells. (A) Transplantation of MCL tumor in a xenografted NSG mouse model. A challenge was performed by transplanting MCL cells into the veins of mice. Tumor cell proliferation was visualized by IVIS imaging 5 days after inoculation of the tumor. Imaging was extended to 120 seconds (day 0) to measure the development of tumor loading. (B) Mice such as (A) were imaged again on day 0 for 10 seconds to track treatment efficiency and reduce bioluminescence intensity for better visibility. On the same day, mice were fed 3 × 10 ⁶ anti-CXCR5 transduced T cells (n = 4) and SP6-CAR transduced T cells (n = 3) were used as negative controls (day 0). IVIS exposure was performed for 10 seconds after the introduction of CAR-T cells to allow a better comparison between days 0 and 19. (C) Mean values of bioluminescent signals obtained from regions of interest covering the entire body of each mouse are plotted for each group at each time point (FIG. 13C). Lymphoma disease was advanced in virtually all mice treated with SP6-CAR and was characterized by strong luminescence signals throughout the bone marrow of the hind limbs, thoracic and abdominal organs, whereas in the CXCR5 CAR treated group. Obviously it wasn't. This provides the first evidence that CXCR5 CAR-T cells have antitumor activity against B-NHL lymphoma in preclinical in vivo studies.

FIG. 14: Co-culture of CAR transduced human T cells with various target cell lines reveals specific T cell activation by distinguishable CXCR5 ⁺ B-NHL and control cell lines. Functional in vitro co-culture and IFN-γELISA (upper figure), IL-2 ELISA (middle figure), TNF-αELISA (lower figure) were performed. The levels of IFN-γ, IL-2, and TNF-α released indicate T cell activation and T cell functionality. T cells without transfection (UT; white bar), T cells expressing CXCR5-CD28 CAR (H28, red bar), T cells expressing CXCR5-4-1BB (HBB1; blue) Rods), T cells expressing CXCR5-CD28 / 4-1BB (H28BB; green bars), SP6 T cells (SP6; gray bars), target cell lines JeKo-1, DOHH-2, SU- It was co-cultured with DHL4, OCI-Ly7, REH, and NCI-H929. The target cells DOHH-2, SU-DHL4, OCI-Ly7, and JeKo-1 are the specific IFN-γ, IL-2, and TNF-α in response to treatment with the CXCR5 CAR-T cells of the present invention. Indicates release.

FIG. 15: Co-culture of CAR-transduced human T cells and CXCR5-negative primary cells of various target human tissues shows that there is no off-target T cell activation, whereas CXCR5 is expressed. The B-NHL cell line JeKo-1 mediates specific T cell activation and functions as a positive control. Functional in vitro co-culture and IFN-γ ELISA were performed. T cells without transfection (UT; white bar), T cells expressing CXCR5-CD28 CAR (H28, red bar), SP6 T cells (SP6; gray bar) are the primary cells, HUVEC. , HUAEC, HA, HN, HPNC, HCoEpiC, Jurkat, a T-ALL cell line, and JeKo-1, a B-NHL cell line. The absence of IFN-γ release indicates the absence of specific T cell activation in the presence of CXCR5-negative primary cells and the CXCR5-negative T-ALL cell line Jurkat. The CXCR5-CD28 CAR-T cells of the present invention exhibit specific IFN-γ release in response to co-culture with JeKo target cells expressing CXCR5.

The present invention will be demonstrated by the examples disclosed below. The examples provide technical support and a more detailed description of non-limiting embodiments that may be preferred.

To demonstrate the functional properties and benefits of the CAR described herein, consider the following examples:
-Co-culture of CAR transduced human T cells with various target cell lines reveals specific T cell activation by identifiable CXCR5 ⁺ cell lines; readings of IFN-γ as effector cytokines from T cells Was a release;
-Cytotoxic assay reveals selective killing of CXCR5 ⁺ cell lines; virtually no killing was seen in CXCR5-negative cell lines (eg, Nalm6 and NCI-H929);
-In vivo experiments used xenograft NSG mouse models, i) function, ii) off-target reactivity, iii) T cell memory, iv) biosafety of adopted CAR-T cells against B-NHL cell lines It is related to generating data about. For B-NHL, the cytolytic capacity of anti-CXCR5 CAR-T cells is compared to the established anti-CD19 CAR-T cell products.

Example 1: Cloning and plasmid preparation:

CAR scFv sequences were synthesized using GeneArt ™ (Gene Synthesis Service). Restriction digestion of the synthesized sequence was performed with NotI and Csil to release the scFv insert from the GeneArt plasmid. The retroviral vector MP71 containing the additional CAR sequence was digested with NotI and Csil to allow the isolated scFv insert to be ligated to the CAR construct within the vector. The digested vector was then dephosphorylated. Fragments were separated by gel electrophoresis (Fig. 8) and purified.

Then, the CAR scFv sequence generated as described above was ligated to a purified vector (50 ng) containing an additional CAR sequence in a ratio of 3: 1. MACH-1 was transformed with this ligation mixture. Control digestion was performed and the resulting mini-preparation was sequenced. MACH-1 was then transformed again with these constructs. A maxi preparation of the MP71-CXCR5-CAR plasmid was prepared.

MP71 is a positive-strand RNA virus. Reverse transcriptase converts the RNA genome of a retrovirus into a DNA copy. DNA is integrated as a provirus at random locations on the target genome. The virus replicates stably as a provirus through cell division.

MP71 contains the following regulatory cis elements:
The LTR (= long terminal repeat) is derived from the mouse myeloproliferative sarcoma virus (MPSV) and contains a promoter. The leader sequence of the vector is derived from mouse embryonic stem cell virus. PRE (= post-transcriptional regulatory element) is originally derived from the Woodchuck hepatitis virus.

MP71 is deactivated. This is because MP71 is no longer capable of replicating because the retroviral gene has been lost. The structural genes were previously separately introduced into two helper plasmids in the packaging cell line. Transfection requires the gene products gag, pol, and env. Infectious virus particles are released into the supernatant of the cell culture. Infectious viral particles can be used for transduction into PBL. A large transduction rate can be achieved with a large expression rate in human PBL.

Example 2: Transfection and transduction:

Day 0: HEK-T (293T) cells or GalV cells are seeded on a 6-well plate for virus production.

Day 1: Transfect 3-plasmid transfection (calcium phosphate transfection) for retrovirus production. According to standard protocols, per well, using DNA of 10μg in 150μl of H ₂ O containing 250mM of CaCl _2. The cells were incubated at 37 ° C. for 6 hours, the medium was changed and the cells were incubated at 37 ° C. for an additional 48 hours.

Cover a 24-well non-tissue culture plate with anti-huCD3 and anti-huCD28 antibodies:

Prepare 0.5 ml of a solution containing anti-CD3 antibody / anti-CD28 antibody in PBS (5 μg / ml anti-CD3, 1 μg / ml anti-CD28) per well. Each well containing 0.5 ml of antibody solution is incubated at 37 ° C. for 2 hours, replaced with a solution containing sterilized 2% BSA (in water) and incubated for 30 minutes (37 ° C.). The BSA solution is removed and the wells are washed with 2 ml PBS.

Purify PBMCs from 40 ml of blood (approximately 2.5 x 10 ⁷ PBMCs):

12.5 ml Ficol gradient medium is prepared in a 2 x 50 ml Falcon tube, blood is diluted with RPMI (+ 100 IU / ml penicillin, streptomycin) to 45 ml, mixed and 22.5 ml blood-medium-. Cover with mixture and centrifuge (20 minutes, 20 ° C., 1800 rpm, RPMI 648, G 17.9). Discard 15 ml of the upper phase. The remaining upper phase is transferred to a new 50 ml falcon tube with a white milky PBMC-containing intermediate phase, filled with RPMI (+100 IU / ml penicillin, streptomycin) until 45 ml and centrifuged. The pellet was resuspended in 45 ml RPMI (+ 100 IU / ml penicillin, streptomycin), centrifuged, the pellet was assembled in 10-20 ml T cell medium and one sample was stained with trypan blue. Cells are counted and cells are added to wells coated with anti-CD3 / anti-CD28 at a concentration of 1-1.5 × 10 ⁶ cells / ml. The rest of the PBMC is centrifuged, suspended in frozen medium and stored in cryotubes at −80 ° C.

Day 3: Transduction of PBL

The virus supernatant is removed from HEK-T cells or GalV cells and filtered (0.45 μm filter). Stimulated PBMCs are treated with 1.0 ml of virus supernatant. Stimulated PBL is treated with 1 ml of virus supernatant and centrifuged in wells coated with CD3 / CD28 (90 minutes, 32 ° C., 800 xg). The final concentration is 100 IU / ml IL-2, or 10 ng / ml IL-7 and 10 ng / ml IL-15, plus 4 μg / ml (8 μl) protamine sulfate.

Day 4: Transduction of PBL

The remaining virus supernatant (4 ° C.) and the second supernatant from HEK-T cells or GalV cells are filtered (0.45 μm). Collect 1 ml of supernatant from PBL. The cytokine concentration is adjusted to 100 IU / ml IL-2 or 10 ng / ml IL-7 and 10 ng / ml IL-15, as well as to 4 μg / ml (8 μl) protamine sulfate. Centrifuge at 800 xg for 90 minutes at 32 ° C. After 4 hours of transduction, PBL is rinsed from a 24-well plate and placed in a T25 cell culture flask. Fresh medium containing IL-2 or IL-7 / IL-15 is added.

7th-13th days: PBL is cultured and T cell medium is treated with fresh IL2 or IL7 / IL15.

Day 13: Finish T cell stimulation.

The PBL culture from the cell culture flask is rinsed, centrifuged and the pellet resuspended in T cell medium (+10 IU / ml IL-2 or 1 ng / ml IL-7 / IL-15).

Day 15: Functional assay

Example 3: In vitro functional test of anti-CXCR5 CAR T cells

Confirmation of CXCR5 CAR expression on the surface of human T cells after transduction with retrovirus:

Evidence of CAR receptor folding and induction is provided in the context of human T cells. It is demonstrated that the retrovirus transduction protocol works.

1) Human peripheral blood leukocytes were purified with a Ficoll gradient. Cells were cultured, stimulated and transduced with a retrovirus as described above.

Following transduction, cells were further cultured in medium containing IL-2 or IL-7 / IL-15 and then the expression of CXCR5-CAR was analyzed.

2) Transduction rate and survival rate were evaluated by flow cytometry (FACS) analysis. To detect expression of CXCR5-CAR, cell staining was performed with an anti-human IgG antibody that selectively recognizes human IgG1 or human IgG4 compartments within the spacer region of the CAR construct. Co-staining of CD3 / CD8 T cells was performed.

The results are shown in FIG.

Identifiable CXCR5 when CAR-transduced human T cells are co-cultured with various target cell lines ⁺ Finding specific T cell activation by B-NHL cell line

The reading used for CAR-T cell activation was the release of IFN-γ as an effector cytokine from T cells.

1) As described in detail above, transduced human T cells were generated using a retrovirus. CXCR5-CD28 CAR receptor variant (H28), CXCR5-4-1BB CAR receptor variant (HBB1), CXCR5-CD28 / 4-1BB CAR receptor variant (H288BB), SP6 negative control were used as T cells. CAR, UT = T cells without transduction.

2) Human T cells transduced with a retrovirus were co-cultured in the presence of listed cell lines or primary cells in a 1: 1 ratio for 18-20 hours.

3) After co-culture, the cell-free culture supernatant was sampled to determine the control level of IFNγ. The maximum release value was induced by stimulating the effector T cells with PMA / ionomycin, and the minimum release value was induced using only T cells.

4) The release of IFN-γ in the supernatant was determined by ELISA.

The results are shown in FIG.

Cytotoxic assay reveals selective killing of CXCR5-positive cell lines: virtually no killing in CXCR5-negative cell lines

A ⁵¹ Cr release assay was used to quantify the activity of cytotoxic T lymphocytes. This assay makes it possible to measure cell lysis of target cells.

1) As described in detail above, transduced human T cells were generated using a retrovirus. CXCR5-CD28 CAR receptor variant (H28) was used in addition to SP6 negative control CAR and UT = non-transduced T cells.

2) Target cells were labeled with ⁵¹ Cr.

3) Next, CAR-T cells and labeled target cells were co-cultured for 4 hours and the ratio of effector cells to target cells was titrated:

Effector to target ratio:
80: 1
0: 1
20: 1
10: 1
5: 1
2.5: 1

4) The cell-free culture supernatant was collected.

5) The supernatant was transferred to a LUMA-scintillation plate, and the released ⁵¹ Cr was measured with a γ scintillation counter. The maximum release value was determined by the target cells lysed by application to the LUMA plate. The minimum release value was determined using only the target cells.

The results are shown in FIG.

Example 4: In vivo experiment in which the evaluation of adopted CAR-T cells against a B-NHL cell line is performed using a xenotransplanted NGS mouse model.

To demonstrate that CAR-T cells containing the various anti-CXCR5-CAR variants described herein have effector activity under in situ conditions, CXCR5 ⁺ B-NHL cell lines were used in NSG mice (NOD. Cg-Prkdc ^scid). It is transplanted to Il2rg ^{tm1 Wjl} / SzJ) via the intravenous route.

The CXCR5 ⁺ B-NHL cell line is one of SU-DHL4 (DLBCL), JEKO-1 (mantle cell lymphoma), JVM3 (CLL), DOHH-2 (FL), OCI-Ly7, SC-1.

Negative control cell lines are included. It is REH (ALL), which is not destroyed by anti-CXCR5 CAR T cells in vitro, but is a target for anti-CD19 CAR.

These mice are particularly suitable for xenotransplantation. This is because it does not have T cells, B cells, or NK cells that reject transplantation while supporting the proliferation of human cells by supplying cytokines. The firefly luciferase gene is also stably transduced into the B-NHL cell line. Application of luciferin to this gene enables the detection of in vivo tumor cells. The exacerbation and distribution of the injected tumor is monitored by bioluminescence imaging using the IVIS system.

Next, when tumor cell proliferation is confirmed by increasing the intensity of the luciferase luminescence signal approximately 5-8 days after inoculation of the tumor cells, human CAR T cells are started from 5 × 10 ⁵ cells per recipient. Up to 5 x 10 ⁶ cells should be administered by intravenous route as in titration. Anti-CXCR5 CAR T cells are compared with anti-CD19 CAR and two suitable negative controls (eg, an unrelated SP6 control and a T cell control without transduction).

Luminescence intensity is measured in mice at intervals of 3-5 days to reveal delayed / progression of growth and loss of signals associated with tumor cells. An observation period of 28 days is adopted. Decreased luminescence intensity and loss of signals associated with tumor cells indicate a therapeutic effect on CAR T cells.

The above experiment was performed by NOD. Cg-Prkdc ^scid Il2rg ^{tm1 Wjl} / SzJ (NSG) mice were used, and in this experiment, 5 × 10 ⁵ mantle cell lymphoma cells transduced into the veins of the mice with the luciferase gene in tandem with GFP. (MCL) JeKo-1 was inoculated (Fig. 13). Next, mice were fed 3 × 10 ⁶ anti-CXCR5 CAR transduced T cells (n = 4), and SP6-CAR transduced T cells (n = 3) were used as negative controls. (Day 0). Mean values of bioluminescence signals were taken from areas of interest covering the entire body of each mouse. Data is plotted at each time point for each group (FIG. 13C). Virtually all mice treated with SP6 CAR had advanced lymphoma disease and were characterized by strong luminescence signals throughout the bone marrow of the hind limbs, thoracic and abdominal organs, whereas the group treated with CXCR5 CAR Obviously it wasn't. This provides the first evidence that CXCR5 CAR-T cells have antitumor activity against B-NHL lymphoma in preclinical in vivo studies.

FIG. 14 shows (1) an alternative CAR component having 4-1BB or CD28 as a co-stimulation component, (2) a third generation CAR having CD28 and 4-1BB as a co-stimulation component, and CAR T cells. The effects of these CARs in co-culture experiments using tumor cell lines are shown. Specific activation of CAR T cells by tumor cells carrying CXCR5 is demonstrated by the release of IFNγ, IL-2, THF-α.

Although CXCR5-CD28 (H28) CAR appears to be most effective, two alternative CARs, namely CXCR5-4-1BB (HBB1) and CXCR5-CD28 / 4-1BB (H28BB), also have distinct specific activity. Shown. It can be seen from FIG. 15 that primary cells from healthy human tissue without CXCR5 (see also the data in FIG. 10B) do not induce specific activity of CXCR5-CD28 CAR-T cells.

Example 5: Clinical approach in a specific patient population

In the human in vivo setting, the present invention is expected to be effective in B-NHL patients with diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocyte leukemia, and mantle cell lymphoma. Patients with the following characteristics will be enrolled in Phase I clinical trials: i) patients with multidrug resistance, ii) patients unsuitable for allogeneic stem cell transplantation, iii) with multiple illnesses and additional chemistry Patients who cannot be treated, iv) Elderly patients who cannot tolerate chemotherapy, v) Patients who are eligible for rescue therapy after the appearance of progressive disease and failure of some other standard therapies, vi) Self Patients with rapidly progressive disease after stem cell transplantation, vii) Patients with rapidly progressing disease after allogeneic stem cell transplantation, viii) As a bridging therapy prior to allogeneic stem cell transplantation, and / Or ix) Current antibody therapy (anti-CD20, rituximab; anti-CD19, oletuzumab; BITE CD19 / CD3, brimatsumomab) or anti-existing variants or variants of CD19 and / or CD20 on the surface of tumor cells. Patients for whom CD19 CAR therapy has lost / prepared down the target structure and is ineffective.

In the human in vivo setting, the present invention is effective in angioimmunoblastic T-cell lymphoma and in various forms of T-cell lymphoma with leukemic dissemination, skin localization, and any other organ dissemination. It is expected that. For such patients, there is no selective treatment other than a general chemotherapy treatment plan. Therefore, patients with the following characteristics will be enrolled in Phase I clinical trials: i) patients with multidrug resistance, ii) patients unsuitable for allogeneic stem cell transplantation, iii) having multiple illnesses and further Patients who cannot receive chemotherapy, iv) Elderly patients who cannot tolerate chemotherapy, v) Patients who are eligible for rescue therapy after the appearance of progressive disease and failure of some other standard therapies, vi) Patients with rapidly progressive disease after autologous stem cell transplantation, vii) Patients with rapid progressive disease after allogeneic stem cell transplantation, viii) As a bridging therapy prior to allogeneic stem cell transplantation ix) As a second-line treatment for patients with progressive disease while receiving one standard chemotherapy.

Claims (16)

i) An extracellular antigen-binding domain containing an antibody or antibody fragment that binds to the CXC chemokine receptor type 5 (CXCR5) protein.
ii) Transmembrane domain and
iii) Chimeric antigen receptor polypeptide (CAR) containing the intracellular domain. The extracellular antigen-binding domain
-Heavy chain complementarity determining regions 1 (H-CDR1), whose sequence matches at least 80% with SEQ ID NO: 1 (GFTFSTSG),
-Heavy strand complementarity determining regions 2 (H-CDR2) whose sequence matches at least 80% with SEQ ID NO: 2 (ISSSSGFV), and-Heavy strand complementarity determining regions 2 (H-CDR2) whose sequence matches at least 80% with SEQ ID NO: 3 (ARCEAAF). 3 (H-CDR3)
Variable heavy chain (VH), including
-Light chain complementarity determining regions 1 (L-CDR1), whose sequence is at least 80% identical to SEQ ID NO: 4 (KSRLSRMGITP),
-Light chain complementarity determining regions 2 (L-CDR2) whose sequence is at least 80% identical to SEQ ID NO: 5 (RMS), and-Light chain complementarity determining regions whose sequence is at least 80% identical to SEQ ID NO: 6 (AQFLEYPPT). 3 (L-CDR3)
The chimeric antigen receptor polypeptide (CAR) according to claim 1, which comprises a variable light chain (VL) comprising. The first or second claim, comprising a VH domain comprising the CDR sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and a VL domain comprising the CDR sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Chimeric antigen receptor polypeptide (CAR). SEQ ID NO: 7:
(However, X1 is G or K; X2 is R or K; X3 is A or S; X4 is N or H; X5 is E or D; X6 is S or A; X7 is S or A; X8 is S or T; X9 is M or L; X10 is R or K; X11 is A or S; X12 is V or I)
With a VH domain whose sequences match at least 80%, preferably at least 85%, or 90%, or 95%, or 100%.
SEQ ID NO: 8:
(However, X1 is S or A; X2 is L or V; X3 is P or S; X4 is S or N; X5 is Q or K; X6 is R or L; X7 is G or E)
The chimeric antigen receptor according to any one of claims 1 to 3, wherein the chimeric antigen receptor comprises at least 80% matching sequence, preferably at least 85%, 90%, 95%, or 100% matching VL domain. Body polypeptide (CAR). A VH domain according to SEQ ID NO: 9 (EVQLVESGGGLVQPGGSLRLSCAASGFTFSTSGMNWFRQAPGKGLEWVSYISSSSGFVYADSVKGRFTISRDNAQNSLYLQMNSLRAEDTAVYYCARSEAAFWGQGTLVTVSS), or SEQ ID NO: 10 (EVQLVESGGGLVQPGKSLKLSCSASGFTFSTSGMHWFRQAPGKGLDWVAYISSSSGFVYADAVKGRFTISRDNAQNTLYLQLNSLKSEDTAIYYCARSEAAFWGQGTLVTVSS),
SEQ ID NO: 11 (DIVLTQSPRSLPVTPGEPASISCRSSKSRLSRMGITPLNWYLQKPGQSPQLLIYRMSNRASGVPDRFSGSGSGTDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK), or SEQ ID NO: 12 and a VL domain according to (DIVLTQAPRSVSVTPGESASISCRSNKSRLSRMGITPLNWYLQKPGKSPQLLIYRMSNLASGVPDRFSGSGSETDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK), chimeric antigen receptor polypeptide according to any one of claims 1 to 4 (CAR). When the CAR is expressed in a gene-modified immune cell, preferably in T lymphocytes, the immune cell is activated by binding to CXCR5 on the surface of the cell expressing CXCR5. This induces cytotoxic activity against the cells expressing CXCR5, wherein the cells expressing CXCR5 are preferably DOHH-2 cell line, OCI-Ly7 cell line, SU-DHL4 cell line. The chimeric antigen receptor polypeptide according to any one of claims 1 to 5, which is a JeKo-1 cell line, a JVM-3 cell line, a MEC-1 cell line, and / or an SC-1 cell line. CAR). -The extracellular antigen-binding domain contains a linker polypeptide located between the VH domain and the VL domain, and the linker is preferably a Whiteow linker (SEQ ID NO: 13: GSTSGSGKPGSGEGSTKG), Gly-Ser linker (SEQ ID NO: 13: GSTSGSGKPGSGEGSTKG). SEQ ID NO: 14: SSGGGGSGGGGGSGGGGS), or selected from linkers whose sequence matches at least 80% with SEQ ID NO: 13 or 14; and / or-a spacer polypeptide located between the extracellular antigen binding domain and the transmembrane domain. Is additionally included, where the spacer is
a. IgG1 spacer (SEQ ID NO: 15; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK)
b. IgG1Δ spacer (SEQ ID NO: 16; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSSLSPGKK)
c. IgG4 (Hi-CH2-CH3) spacer (SEQ ID NO: 17; ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK);
d. IgG4 (Hi-CH3) spacer (SEQ ID NO: 18; ESKYGPPPCPPCPGQPREPQVYTLPPPSQEEMTKKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFCSVMSHEALHSLHKSL)
e. IgG4 (Hi) spacer (SEQ ID NO: 19; ESKYGPPCCPPCP); or f. Selected from spacers whose sequence matches at least 80% with any one of SEQ ID NOs: 15-19, and / or-the transmembrane domain is the CD8α domain (SEQ ID NO: 20; IYIWAPLAGTCGGVLLSLVITLYC), CD28 domain (SEQ ID NO: 21; FWVLVVVGVGVLACYSLLVTVAFIIFWV), or transmembrane domain whose sequence matches at least 80% with SEQ ID NO: 20 or 21; and / or-the intracellular domain is the 4-1BB co-stimulating domain (SEQ ID NO: 22; KRGRKKLLYIFKQPFMRRPVQTTQCRFPEEG28) A co-stimulation domain containing both the co-stimulation domain (SEQ ID NO: 23; RSKRSRLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSL), 4-1BB (SEQ ID NO: 22) and the CD28 co-stimulation domain (SEQ ID NO: 23) in adjacent arrangements, or a sequence with SEQ ID NO: 22 or 23. Includes a co-stimulation domain selected from at least 80% matching co-stimulation domains; and / or additionally contains an additional signal transduction domain (activation domain), wherein the signal transduction domain is CD3ζ (4-1BB or CD28). Signal transduction domain (SEQ ID NO: 24; LRVKFSRASADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMMKGERRRRGKGHDGLYQGLSTATKDTYDALHQ at least sequence number 24
The chimeric antigen receptor polypeptide (CAR) according to any one of claims 1 to 6. The chimeric antigen receptor polypeptide (CAR) according to any one of claims 1 to 7, which comprises or comprises a sequence according to any one of SEQ ID NOs: 25, 26, 27, 28, 29. An isolated nucleic acid molecule, preferably in the form of a vector, such as a viral vector or a transposon vector, preferably a sleeping beauty vector.
a) Below:
− Encoding the chimeric antigen receptor (CAR) polypeptide according to any one of claims 1-8.
-It encodes an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain is composed of at least one sequence of SEQ ID NO: 37, 53 or 38 and SEQ ID NO: 39, 54 or Nucleic acid molecule encoded by at least one of 40 or containing a nucleotide sequence according to SEQ ID NO: 31, 32, 33, 34, or 35; and / or complementary to the nucleotide sequence set forth in b) a). Nucleic acid molecule;
c) A nucleotide sequence having a sequence that matches the nucleotide sequence of a) or b) to the extent that it has a similar / equivalent function, preferably the nucleotide sequence and sequence of a) or b). Nucleic acid molecules containing nucleotide sequences that match at least 80%;
d) Nucleic acid molecules that result in the nucleotide sequences described in a) to c) by degeneration; and / or e) modified by deletion, addition, substitution, transposition, reversal, and / or insertion as a consequence of the genetic code. However, an isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules according to the nucleotide sequences according to a) to d), which have similar / equivalent functions to the nucleotide sequences described in a) to d). A genetically modified immune cell comprising the nucleic acid molecule of claim 9 and / or expressing the CAR of any one of claims 1-8. The genetically modified immune cell according to claim 10, wherein the immune cell is selected from the group consisting of T lymphocytes and NK cells, wherein the T lymphocytes are preferably cytotoxic T lymphocytes. For the treatment of medical disorders associated with the presence of pathogenic cells expressing CXCR5, preferably pathogenic mature B cells and / or memory B cells, and / or pathogenic T cells and / or T follicular helper cells. The genetically modified immune cell according to claim 10 or 11, for use. The immune cell according to claim 12, wherein the medical disorder is a mature B cell non-Hodgkin's lymphoma (B-NHL) for use as a drug. The immune cell according to claim 12, wherein the medical disorder is a T-cell non-Hodgkin's lymphoma with or without leukemia tumor cell dissemination. The medical disorder
-From a group consisting of acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and diffuse large B cell lymphoma (DLBCL) B-cell-derived lymphoproliferative disorders of choice; or-T-cell-derived lymphoproliferative disorders selected from the group consisting of vascular immunoblastic T-cell lymphoma, cutaneous T-cell lymphoma, and T-cell lymphoma with leukemic dissemination. The immune cell according to claim 12, which is used as a drug. The immune cell according to claim 12, wherein the medical disorder is an autoantibody-dependent autoimmune disease, preferably selected from systemic lupus erythematosus (SLE) or rheumatoid arthritis, for use as a drug.